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Objective: We analyzed the gestational changes of pharmacological activity and molecular levels of KATP channels in rat myometrium., Study Design: Using rat myometrium, the effects of K+ channel openers (KCOs) were examined in an isometric tension study of oxytocin-induced contraction. We also examined the effects of KCOs on the intracellular Ca2+ levels of cultured myometrial cells. The expression of myometrial KATP channels was assessed by RT-PCR and Northern blot analysis., Results: The effect of KCOs were altered during pregnancy, with a significant increase of their potency at day 18 of pregnancy followed by a decline towards the non-pregnant level at the day of delivery. KCOs suppressed the Ca2+ influx across the cell membrane. The mRNAs encoding each component of myometrial KATP channels, Kir6.1 and SUR2B, exhibited gestational stage-dependent alterations similar to those of the effects of KCOs., Conclusion: These findings suggest that KCOs inhibit uterine myometrial contraction more effectively during pregnancy than in the non-pregnant state due to gestation-enhanced expression of KATP channels, implying that KCOs might be useful for preventing premature delivery.

Objective: The development and maturation of the ovarian follicles are characterized by structural changes that require components involved in cell-cell adhesion systems. Recently a novel group of cell adhesion molecules named nectins has been identified. The present study examined expression and cell-specific localization of nectins during mouse follicular development., Study Design: Expression of nectins in mouse ovary was investigated by immnuoblot analysis and immunohistochemistry. More precise localization was determined by electron microscopy., Results: Immunoblot analysis revealed expression of nectin-2 and nectin-3 but not nectin-1 in ovarian granulosa cells. Immunohistochemistry demonstrated expression of nectin-2 at cell-cell adhesion sites of granulosa cell layer in the primary and preantral follicles. Especially, intense immunoreactivity of nectin-2 accumulated around the zona pellucida. In antral follicles, the intensity of nectin-2 expression on granulosa cells was decreased. By electron microscopy nectin-2 was detected not only on thin extensions of granulosa cells penetrating the zona pellucida, but also on the attachment sites between thin extensions of granulosa cells and oocyte surface., Conclusion: The restricted expression of nectin-2 in the granulosa cells of primary and preantral follicles might reflect some of the molecular changes in cell-cell adhesion during early follicular development.

HMG-CoA reductase inhibitors and calcium channel blockers have antiatherogenic effects; however, their mechanisms remain to be elucidated. This study examined the effect of cerivastatin and/or nifedipine on the endothelial dysfunction in porcine balloon-injured coronary arteries. Normal male pigs were randomly divided into the following four groups: control, cerivastatin (1 mg/kg/d PO), nifedipine (4 mg/kg/d PO), and their combination (n = 10 each). We started the treatments 3 days before balloon injury in the proximal left coronary arteries and continued for 4 weeks after the procedure. Then, we examined endothelial vasodilator functions ex vivo in organ chambers and in vitro by Western blotting for eNOS expression. Endothelium-dependent relaxations to serotonin, but not those to bradykinin or the calcium ionophore A23187 or endothelium-independent relaxations to sodium nitroprusside, were significantly impaired by balloon injury. The monotherapy with cerivastatin or nifedipine partially improved, and their combination supernormalized the relaxations to serotonin without affecting those to bradykinin or A23187 or endothelium-independent relaxations to sodium nitroprusside. The expression of eNOS was significantly reduced by balloon injury and normalized by the combination therapy. These results indicate that the combination therapy improves endothelial dysfunction after balloon injury, in which the up-regulation of eNOS may be involved.

Background: Lysophosphatidic acid (LPA) induced a dose-dependent increase of cancer cell invasion by promoting Rho/Rho-associated kinase signaling. Prenylation of Rho is essential for regulating cell growth, motility, and invasion. Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an antiulcer drug. Recent findings suggested that GGA might inhibit the small GTPase activation by suppressing prenylation. The authors hypothesized that the anticancer effects of GGA result from the inhibition of Rho activation., Methods: The authors examined the effect of GGA using an in vitro invasion assay in human ovarian carcinoma cells, and analyzed the mechanism of the GGA effect on Rho activation, stress fiber formation and focal adhesion assembly, which are essential processes for cell invasion., Results: The induction of ovarian carcinoma cell invasion by LPA was inhibited by the addition of GGA in a dose-dependent manner. Treatment of cancer cells with GGA resulted in inactivation of Rho, changes in cell morphology, loss of stress fiber formation and focal adhesion assembly, and the suppression of tyrosine phosphorylation of focal adhesion proteins. The effect of GGA on cancer cells was partially prevented by the addition of geranylgeraniol, which is an intermediate of geranylgeranyl pyrophosphate and compensates geranylgeranylation of Rho., Conclusions: The inhibition of LPA-induced invasion by GGA was, at least in part, derived from suppressed Rho activation by preventing geranylgeranylation., (Copyright 2005 American Cancer Society.)

Objective: The small guanosine triphosphatase RhoA/Rho-kinase cascade has been implicated in uterine contraction. Our purpose was to evaluate the tocolytic effect of a Rho-kinase inhibitor, Y-27632, in lipopolysaccharide-induced preterm delivery in mice., Study Design: We used an animal model of lipopolysaccharide-induced preterm delivery in C3H/HeN x B6D2F1 pregnant mice. Y-27632 was delivered continuously through an osmotic pump that was implanted into the peritoneal cavity 6 hours before lipopolysaccharide treatment. The primary outcome was the preterm delivery rate. To further study the possible involvement of this cascade in lipopolysaccharide-induced preterm delivery, we determined the effect of lipopolysaccharide and prostaglandin F2alpha on RhoA activation in mouse myometrial cells and uterine smooth muscle tissues., Results: The rate of preterm delivery for lipopolysaccharide-treated animals was 94.4%. The administration of Y-27632 (1 or 10 mg/kg/d) significantly reduced the preterm delivery rate to 61.1% or 15.8%, respectively. The level of guanosine triphosphate-bound RhoA was increased after the addition of lipopolysaccharide or prostaglandin F2alpha., Conclusion: The RhoA/Rho-kinase cascade is involved in lipopolysaccharide-induced preterm delivery, which suggests that Rho-kinase could be used as a new therapeutic target for the prevention of preterm labor.

To examine the reason for the formation and the structure of cubic ice in a restricted space, we measured the powder x-ray diffraction patterns of cubic ice formed within the mesopores of porous silicas as a function of pore size (4-70 nm). The results strongly suggest that cubic ice formed in the mesopores does not take a cubic structure as envisaged by Konig. It may be actually composed of very small crystallites of hexagonal ice that contains a large amount of growth faults depending on the crystallite size, that is, ice with disordered stacking sequence. Suppression of crystal growth of ice in the mesopores seems to be a vital factor for the formation and the stability of cubic ice., ((c) 2005 American Institute of Physics.)

Ovarian cancer is characterized by diffuse peritoneal carcinomatosis and often by large volumes of ascites. We previously reported that alendronate, a nitrogen-containing bisphosphonate, inhibited ovarian cancer cell migration by attenuating the activation of Rho through inhibiting the mevalonate pathway. However, questions remain about the ability of alendronate to inhibit the invasiveness of cancer cells to the adherent tissues and the growth of disseminated ovarian cancer in vivo. We established an in vivo ovarian cancer model with i.p. carcinomatosis in athymic immunodeficient mice. In the prevention model, in which alendronate administration started from the day after tumor inoculation, alendronate prevented the stromal invasion, reduced the tumor burden, and inhibited ascites accumulation. Histologic observation revealed that alendronate treatment decreased the stromal invasion of the i.p. tumor while inhibiting the metalloproteinase-2 activity in ascites. This antitumor effect might result from the inhibition of cancer cell migration and proteolytic activity. In the treatment model, in which alendronate was given from 10 days after tumor inoculation when macroscopic tumors are already implanted in the peritoneum, the antitumor effect was weaker but still significant. Furthermore, alendronate administration decreased the serum CA-125 levels of mice bearing disseminated ovarian cancer compared with those of nontreated mice. The potent effects of alendronate in reducing stromal invasion, tumor burden, and ascites suggest that it will be of value in regimens for treatment of women with ovarian cancer.

The mouse blastocyst consists of the trophectoderm, the inner cell mass, and a fluid-filled cavity, the blastocoel. Formation and subsequent expansion of this cavity is important for further differentiation of the inner cell mass and successful implantation. Previous work provided evidence that vectorial transport of Na+ and CL- ions through the trophectoderm into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. As the activity of the Na+ / H+ exchanger (NHE) has been implicated as the exchanger responsible for facilitating the transtrophectodermal Na+ flux, the functional role of NHE in mouse blastocoel development was determined. Embryos were cultured in the presence of subtype-specific NHE inhibitors to examine the role of NHEs in blastocoel development. When 2-cell stage embryos were treated continuously with a specific inhibitor of NHE-1, cariporide, the embryos passed beyond the 8-cell stage and became blastocysts. However, in the presence of a specific inhibitor of NHE-3, S3226, the 2-cell stage embryos developed to the morula stage but formation of the blastocyst were inhibited in a dose-dependent manner. Cariporide did not inhibit the formation of the blastocoel cavity from the morula stage whereas S3226 did inhibit that process. S3226 also reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. An immunofluorescence study showed that NHE-3 was detected in the vicinity of the cell membrane of the trophectoderm, especially in the apical cell margins of the trophectoderm. These results suggest that NHE-3 is likely involved in blastocyst formation.

During mammalian embryonic development, the formation and subsequent expansion of a fluid-filled cavity, the blastocoel, is crucial for successful implantation. Our present experiments were aimed at exploring the contribution of Rho-kinase, a downstream effector of the small GTP-binding protein RhoA, to mouse blastocoel formation. RT-PCR analysis showed that Rho-kinase mRNA is present throughout mouse preimplantation development. When 2-cell embryos were cultured in the presence of a specific inhibitor of Rho-kinase, Y-27632, they developed to the morula stage but failed to develop to the blastocyst stage. Y-27632 inhibited the formation of the blastocoel cavity from the morula stage, and this inhibitory effect was reversible when embryos were returned to medium without Y-27632. Moreover, Y-27632 reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. These results suggest that Rho-kinase is likely involved in blastocyst formation.

Objective: We recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is substantially involved in the pathogenesis of arteriosclerosis. In this study, we examined whether Rho-kinase is also involved in in-stent restenosis and if so, what mechanism is involved., Methods and Results: Pigs underwent stent implantation in the left coronary artery with or without administration of fasudil (30 mg/kg per day orally), a specific Rho-kinase inhibitor, starting 2 days before the procedure for a duration of 4 weeks. On day 28, reductions in coronary diameter and neointimal formation associated with macrophage accumulation, collagen deposition, and transforming growth factor (TGF)-beta1 expression were noted at the stent site, and all were significantly suppressed by fasudil. On day 7, fasudil significantly increased the frequency of TUNEL-positive apoptotic cells, while it tended to reduce that of bromodeoxyuridine-positive proliferating cells in the neointima. Western blot analysis on day 7 demonstrated that phosphorylations of the ezrin/radixin/moesin family (a marker of Rho-kinase activity in vivo) and protein expression of monocyte chemoattractant protein-1and bcl-2 were upregulated at the stent site and were significantly suppressed by fasudil., Conclusions: These results indicate that long-term inhibition of Rho-kinase suppresses in-stent neointimal formation by multiple mechanisms, including reduced vascular inflammation, enhanced apoptosis, and decreased collagen deposition.

The trophoblast, an important component of the mammalian placenta, has several essential biological roles in the maintenance of pregnancy. First, trophoblast cells must attach to the uterine endometrium, and then they must invade to a depth at which the vascular network exists. Here, we investigated the effects of epidermal growth factor (EGF) on alpha2 integrin expression, adhesiveness to collagen, and invasive activity using human BeWo choriocarcinoma cells. EGF induced the expression of alpha2 integrin mRNA and protein, as shown by Northern blotting, Western blotting, and flow cytometry. Human chorionic gonadotropin (hCG) secretion was enhanced by the addition of EGF, which suggests that the BeWo cells functionally differentiated similarly to normal trophoblasts. EGF also dose-dependently stimulated the invasiveness of BeWo cells. Antibody against alpha2 integrin inhibited this effect, suggesting that it may be mediated by an increase of cell surface integrin. EGF had no effect on the adhesiveness of BeWo cells to collagen, whereas it stimulated the chemokinetic activity in a dose-dependent manner. The increase of chemokinetic activity was suppressed by antibody against alpha2 integrin. These results suggest that EGF may induce alpha2 integrin expression in trophoblast cells, thereby enhancing their invasiveness into the endometrium via an increase of their chemokinetic activity.

Objective: We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm., Methods and Results: A segment of left porcine coronary artery was chronically treated with IL-1beta-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1beta-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTPgammaS-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKCdelta isoform was activated during the hypercontraction., Conclusions: These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKCdelta may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

Hormone replacement therapy (HRT) is effective treatment for postmenopausal bone loss and osteoporosis. However, postmenopausal women with low bone mass fail to respond after a few years of HRT and some postmenopausal women show no bone response to HRT. In these cases, additive therapy is necessary. Bisphosphonates are highly effective in postmenopausal osteoporosis. We investigated the effects of adding etidronate, a bisphosphonate, to HRT. The addition of etidronate to HRT improved bone density (4.40+/-1.12% increase per year) in patients showing no increase or even a decrease after at least 1 year of HRT (5.39+/-2.40% decrease per year).

Although the importance of monocytes/macrophages in the pathogenesis of arteriosclerosis is widely accepted, effective and safe treatment to inhibit those inflammatory cells remains to be developed. It was recently found that propagermanium, which is clinically used for the treatment of chronic hepatitis type B in Japan, markedly suppresses monocyte chemotaxis in response to macrophage chemoattractant protein-1 (MCP-1) through inhibition of its receptor, C-C chemokine receptor 2, in vitro. This prompted examination of whether propagermanium suppresses the macrophage-mediated formation of coronary arteriosclerotic lesions in our porcine model in vivo. It was first confirmed that propagermanium inhibited the migration of porcine monocytes in response to MCP-1 at therapeutic concentrations in vitro. Pigs were randomly divided into two groups; one group was orally treated with propagermanium (1 mg/kg, three times/day) and another group served as a control (n = 6 each). Porcine coronary segment was treated from the adventitia with MCP-1 and oxidized low-density lipoprotein for 2 weeks. In the control group, this treatment resulted in the development of stenotic coronary lesions with hyperconstrictive responses to serotonin where arteriosclerotic lesions (neointimal formation and constrictive remodeling) were developed. Immunohistochemical analysis demonstrated the macrophage accumulation in the adventitia and the media. By contrast, in the propagermanium group, angiographic coronary stenosis, hyperconstrictive responses, histologic changes, and macrophage accumulation were all significantly suppressed. These results indicate that propagermanium suppresses macrophage-mediated formation of coronary arteriosclerotic lesions in vivo, suggesting its potential usefulness for the treatment of arteriosclerotic vascular diseases.

Objectives: Estrogen acts directly on endothelial nitric oxide synthase through a non-genomic mechanism, resulting in rapid dilatation of blood vessels. In this study, we examined the change of endothelial function after surgical menopause., Methods: In 20 subjects who underwent gynecological operations (ovariectomy (OVX) 12, sham (SHAM) operation 8), postoperative changes of flow-mediated dilatation (FMD) of the brachial artery were examined using ultrasonography. Postoperative changes of the response to nitroglycerin (NTG) were also studied in these patients., Results: In the OVX group, significant decreases of FMD were observed 1 week after the operation, although no changes were observed in the response to NTG. In the SHAM group, no remarkable changes of FMD or the response to NTG were observed after the operation., Conclusions: OVX influences endothelium-dependent vasodilatation within as little as 1 week. Therefore, it may be important to address the rapid changes of circulation after surgical menopause in order to prevent cardiovascular disease.

Macrophages play an important role in the pathogenesis of atherosclerosis, for which monocyte chemoattractant protein (MCP)-1 and CCR2 chemokine receptors may be involved. The authors have recently demonstrated that propagermanium exerts inhibitory effect on the CCR2 receptors. In the current study, the authors examined whether the organic germanium suppresses the MCP-1-induced monocyte migration in vitro and the development of atherosclerosis in WHHL rabbits in vivo. In the in vitro experiment, propagermanium concentration-dependently suppressed the MCP-1-induced migration of THP-1 cells. In the in vivo experiment, 20 WHHL rabbits were randomly divided into two groups; one group was treated with oral administration with propagermanium (9 mg/kg/day) for 3 months, and another group served as a control (n = 10 each). After 3 months, the aorta was isolated and stained with oil red O staining, and neointimal formation was quantified. Macrophage accumulation in the aorta was also evaluated by immunostaining. Long-term treatment with propagermanium did not affect the serum lipid profiles. However, the treatment significantly suppressed the oil red O-positive area of the total aorta (p < 0.05). Similarly, propagermanium significantly suppressed the intimal lesions (maximal intimal thickness and intimal area) and macrophage staining-positive area (all p < 0.05). A significant positive correlation was noted between macrophage staining-positive area and intimal lesions (p < 0.0001). These results indicate that long-term treatment with propagermanium suppresses the development of atherosclerosis in WHHL rabbits, suggesting its usefulness for the treatment of atherosclerotic vascular disease in humans.

Objective: This study was designed to examine why in WHHL rabbits, muscular arteries, such as the carotid artery, are relatively resistant to atherosclerosis compared with the aorta, with a special reference to monocyte chemoattractant protein (MCP)-1., Methods and Results: MCP-1 mRNA expression was quantitated by Northern blot analysis, and its protein expression was quantitated by immunostaining and ELISA at the age of 1, 3, 6, and 12 months (n=5 to 6 each). In the aorta, atherosclerotic lesions were progressively developed with aging, and MCP-1 was highly expressed in endothelial cells and infiltrating macrophages. By contrast, in the carotid artery, atherosclerotic lesions and MCP-1 immunoreactivity were not evident throughout the experimental period. Unexpectedly, however, the extent of MCP-1 mRNA expression was comparable between the aorta and the carotid artery throughout the experimental period. Endothelial cells in primary culture from the aorta and the carotid artery expressed the same extent of MCP-1 mRNA on stimulation by oxidized LDL. There was no abnormality in primary structure of MCP-1 cDNA in WHHL., Conclusions: These results suggest that in WHHL, the atherosclerosis process, including MCP-1 protein expression, may be reduced in the carotid artery (and possibly in other muscular arteries), accounting in part for the regional resistance to atherosclerosis.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other nitrogen-containing bisphosphonates inhibit the mevalonate pathway and thereby inhibit the synthesis of products derived from this metabolite. This, in turn, prevents the prenylation of a number of small GTPases, which regulate cell growth, motility, and invasion. We studied the effect of alendronate on in vitro migration of human ovarian cancer cells. Lysophosphatidic acid (LPA) induced a dose-dependent increase of migration of cancer cells by promoting Rho/Rho-associated kinase signaling. The induction of cancer cell migration by LPA was inhibited by the addition of alendronate in a dose-dependent manner. Treatment of ovarian cancer cells with alendronate resulted in inactivation of Rho, changes of cell morphology, loss of stress fiber formation, and focal adhesion assembly, and the suppression of phosphorylation of myosin light chain and tyrosine phosphorylation of focal adhesion proteins, which are essential processes for cell migration. The effects of alendronate on cancer cells were prevented by the addition of geranylgeranyol, which is the metabolic intermediate of the mevalonate pathway. These results suggest that alendronate inhibits Rho activation by preventing geranylgeranylation, which results in inhibition of LPA-induced migration of human ovarian cancer cells.

Recent studies demonstrated that neointimal formation, which is caused by both neointimal proliferation and organized mural thrombus, is responsible for in-stent restenosis. Although various types of heparin coatings were effective in reducing (sub)acute thrombosis, most of them failed to reduce neointimal proliferation. This study was designed to examine the effect of the stent coated with multiple layers of releasable heparin complex from which heparin diffuses into the surrounding tissue and exerts its beneficial effects. Male Yorkshire pigs underwent balloon expandable stenting for coronary segments of both the left anterior and the left circumflex coronary arteries with a comparable diameter (n = 10). The stent implantation site was randomized for either control or heparin-coated stent. Four weeks after the procedure, quantitative coronary angiography (QCA) and intravascular ultrasonographic imaging (IVUS) were performed followed by histologic analysis. In additional animals, staining for proliferating cell nuclear antigen (PCNA) was performed 10 d after the procedure (n = 3). QCA demonstrated that coronary diameter (mm) was significantly larger at the heparin-coated stent site (2.32 +/- 0.14) compared with the control stent site (1.81 +/- 0.17) (p < 0.01). IVUS also showed that the neointimal area (mm2) was significantly suppressed at the heparin-coated stent site (2.12 +/- 0.58) compared with the control stent site (3.92 +/- 0.33) (p < 0.01). Histologic analysis also demonstrated that neointimal area (mm2) was significantly less at the heparin-coated stent (2.94 +/- 0.43) than at the control stent site (4.41 +/- 0.38) (p < 0.01), which was also the case for organized thrombus area (x10-4 mm2) (6.61 +/- 2.67 vs. 19.36 +/- 4.38, p < 0.01). The frequency of PCNA-positive vascular smooth muscle cells (%) was significantly less at the heparin-coated stent (10.8 +/- 1.0) than at the control stent site (19.1 +/- 1.7) (p < 0.01). These results suggest that the stent coated with releasable heparin is beneficial in reducing neointimal formation and subsequent in-stent restenosis.

The RhoA/Rho-kinase cascade is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. We examined the potential role of this pathway in oxytocin-induced uterine contraction. The specific Rho-kinase inhibitor Y-27632 inhibited oxytocin-induced rat uterine contraction on d 21 of pregnancy in a concentration-dependent manner, whereas the extent of this inhibition was reduced in the nonpregnant uterus. Y-27632 had no effect on oxytocin-induced intracellular Ca(2+) mobilization in myometrial cells. Immunoblot analysis showed that oxytocin increased the level of myosin light chain phosphorylation, and this increase was attenuated by Y-27632. Oxytocin increased the phosphorylation of myosin-binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase, and this increase was reduced by Y-27632. The expression of Rho-kinase protein was shown to increase in the uterus during pregnancy compared with the nonpregnant uterus, whereas the expression of RhoA protein remained at the same level during pregnancy. RT-PCR and Northern blot analysis showed that the expression of Rho-kinase was up-regulated at the transcriptional level during pregnancy. These results suggest that the RhoA/Rho-kinase pathway may have an important role in oxytocin-induced uterine contraction, and that up-regulation of Rho-kinase is involved in the mechanism underlying the increased contractility of the pregnant myometrium.

Small GTPase Rho and its target Rho-kinase play an important role in various cellular functions that may be involved in the pathogenesis of arteriosclerosis. Here we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (AdDNRhoK) induces a regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1,beta which resulted in the development of constrictive remodeling and vasospastic responses to serotonin in vivo. AdDNRhoK, but not that of beta-galactosidase, into the interleukin-1beta-treated coronary segment caused regression of constrictive remodeling and abolished vasospastic activity in 3 weeks. The unregulated phosphorylation of the target proteins of Rho-kinase at the coronary lesion was significantly suppressed by AdDNRhoK. These results indicate that Rho-kinase is substantially involved in the mechanism of coronary arteriosclerosis, which can be reversed by selective inhibition of the molecule in our porcine model in vivo.

Objective: We recently demonstrated that Rho-kinase/ROK/ROCK is functionally upregulated at the arteriosclerotic coronary lesions and plays a key role for coronary vasospastic responses in our porcine model with interleukin (IL)-1beta. In the present study, we tested our hypothesis that Rho-kinase is involved in the pathogenesis of coronary arteriosclerosis per se in our porcine model., Methods: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1beta-treated site, as previously reported. Then, animals were randomly divided into two groups; one group was treated with fasudil for 8 weeks followed by 1 or 4 weeks of washout period and another group served as a control. After oral absorption, fasudil is metabolized to hydroxyfasudil that is a specific inhibitor of Rho-kinase., Results: In the fasudil group, coronary stenosis and vasospastic response were progressively reduced in vivo, while the coronary hyperreactivity was abolished both in vivo and in vitro. Furthermore, Western blot analysis showed that in the fasudil group, the Rho-kinase activity (as evaluated by the extent of phosphorylation of myosin binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase) was significantly reduced, while histological examination demonstrated a marked regression of the coronary constrictive remodeling., Conclusions: These results indicate that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by long-term inhibition of the molecule in vivo. Thus, Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular disease.

Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1beta, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of beta-galactosidase, into the interleukin-1beta-treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRHOK: These results indicate that Rho-kinase is substantially involved in coronary constrictive remodeling and vasospastic responses, both of which can be reversed by the selective inhibition of the molecule in our porcine model in vivo.

We have previously shown that long-term treatment with an inflammatory cytokine from the adventitia causes the development of coronary vascular lesions, with the accumulation of macrophages. Recent studies in vitro have suggested that small G-protein Rho and its effector, Rho-kinase/ROK/ROCK, may be the key molecules for various cellular functions, including cell adhesion and movement. In this study, we examined whether adventitia-derived macrophages cause the formation of coronary vascular lesions in vivo and, if so, whether Rho-kinase is involved in the process. Porcine coronary segments from the adventitia were chronically treated with monocyte chemoattractant protein-1 alone, oxidized low density lipoprotein alone, or both. Vascular lesion formation (neointimal formation and development of vascular remodeling) was mostly enhanced at the coronary segment cotreated with monocyte chemoattractant protein-1 and oxidized low density lipoprotein, where the phosphorylation of myosin binding subunit of myosin phosphatase was increased, indicating an increased activity of Rho-kinase in vivo. Histological examination demonstrated that macrophages were accumulated at the adventitia and thereafter migrated into the vascular wall. Long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor (hydroxyfasudil) after oral absorption, markedly inhibited the myosin binding subunit phosphorylation, the macrophage accumulation and migration, and the coronary lesion formation in vivo. These results indicate that Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in our porcine model in vivo.

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or beta-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.

Objective: This study was designed to examine whether or not adenovirus-mediated gene transfer of C-type natriuretic peptide (CNP) can prevent coronary restenotic changes after balloon injury in pigs in vivo., Background: Gene therapy to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) might be useful but requires a method applicable for in vivo gene delivery into the coronary artery as well as the efficient vector encoding a potent antiproliferative substance. We tested whether the adenovirus-mediated gene transfer of CNP by use of an infiltrator angioplasty balloon catheter (IABC) might prevent the coronary restenotic changes after balloon injury., Methods: Balloon angioplasty was performed in the left anterior descending and the left circumflex coronary artery in pigs. Immediately after the balloon injury, adenovirus solution encoding either CNP (AdCACNP) or beta-galactosidase (AdCALacZ) gene was injected with IABC into the balloon-injured coronary segments. Expression of CNP was assessed by immunohistochemical staining and cyclic guanosine 3',5'-monophosphate (cGMP) measurement. Coronary restenotic changes were evaluated by both angiographic and histological examinations., Results: CNP was highly expressed in the media and the adventitia of the coronary artery at the AdCACNP-transfected but not at the AdCALacZ-transfected segment. In the AdCALacZ-transfected segment, vascular cGMP levels tended to be reduced as compared with the untreated segment, whereas in the AdCACNP-transfected segment, vascular cGMP levels were restored. Angiographic coronary stenosis was significantly less at the AdCACNP-transfected than at the AdCALacZ-transfected segment. Histological examination revealed that this was achieved primarily by the marked inhibition of the geometric remodeling of the coronary artery by the CNP gene transfer., Conclusions: Adenovirus-mediated CNP gene transfer with the IABC system may be a useful gene therapy to prevent restenosis after PTCA in vivo.

The beta phase of Cu1.75Se has an anti-fluorite structure, where one in eight tetrahedral copper sites are vacant. Powder X-ray diffraction measurements on Cu1.75Se showed that the beta phase transformed to a two-phase mixture of (alpha + beta) at approximately 250 K, and then the beta phase changed to a new phase at approximately 180 K. Powder X-ray and electron diffraction measurements revealed that the low-temperature phase of the beta phase has a superstructure of (2alpha(fcc) x 2alpha(fcc) x 2alpha(fcc)) type, where alpha(fcc) is the lattice parameter of the original beta phase. The superstructure was interpreted to originate from the ordering copper vacancies. The new phase was referred to as the beta' phase.

Stimulation-regulated fusion of vesicles to the plasma membrane is an essential step for hormone secretion but may also serve for the recruitment of functional proteins to the plasma membrane. While studying the distribution of G protein-gated K+ (KG) channels in the anterior pituitary lobe, we found KG channel subunits Kir3.1 and Kir3.4 localized on the membranes of intracellular dense core vesicles that contained thyrotropin. Stimulation of these thyrotroph cells with thyrotropin-releasing hormone provoked fusion of vesicles to the plasma membrane, increased expression of Kir3.1 and Kir3.4 subunits in the plasma membrane, and markedly enhanced KG currents stimulated by dopamine and somatostatin. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for hormone secretion in the endocrine system.

G-protein-gated K+ (KG) channels generate slow inhibitory postsynaptic potentials in the brain. Current opinion suggests that neuronal KG channels are heterotetramers of Kir3.1 and Kir3.2. In substantia nigra (SN), however, mRNA of Kir3.1 does not express, whereas that of Kir3.2 clearly does. Therefore, we have characterized the KG channels containing Kir3.2 subunits in SN using biochemical and immunological techniques. We found that they were composed of only Kir3.2 subunits and did not contain significant amounts of either Kir3.1 or Kir3.3. Furthermore, at least some of the KG channels in SN were assemblies of the splicing variants Kir3. 2a and Kir3.2c. The channels were localized specifically at the postsynaptic membrane on the dendrites of dopaminergic neurons. Kir3. 2c, but not Kir3.2a, could bind a PDZ domain-containing protein, PSD-95. The heterologously expressed KG channels composed of Kir3.2a plus Kir3.2c or Kir3.2a alone were activated by G-protein stimulation, but expression of Kir3.2c alone was not. This study reveals that the Kir3.2 splicing variants play distinct roles in the control of function and localization of some of the KG channels in dopaminergic neurons of SN.

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

Progestogen suppresses the progression of endometrial cancer and has an important effect on the secretory change of human endometrium. We characterized the progestogen-induced alterations of gene expression in a human endometrial-cancer cell line using a mRNA differential-display reverse-transcriptase-polymerase-chain-reaction (DDRT-PCR) method. After 5-day incubation of Ishikawa endometrial-cancer cells, with or without 100 nM medroxyprogesterone acetate (M PA), total RNA was isolated from confluent cells. We identified 8 candidate genes by mRNA differential display by screening up to approximately 3,000 mRNA species. Among these, 2 genes named T21A and T21B showed a decrease in mRNA by MPA treatment when analyzed by Northern blot. Nucleotide sequence showed that clone T21A was part of human mitochondrial short-chain enoyl-CoA hydratase cDNA. The other clone, T21B, showed no homology with any known nucleotide sequences. Northern-blot analysis using T21A and T21B clones as probes showed a decrease in mRNA in human endometrium from the luteal stage, with high serum estradiol and progesterone levels, as compared with that from the early follicular stage, with low serum estradiol and progesterone levels, and that from the pre-ovulatory stage with high serum estradiol and low progesterone levels. These findings suggest that mRNA DDRT-PCR could be used to identify the candidate genes regulated by progestogen in human endometrial cancer and in normal human endometrium.

The purpose of this study was to analyze the mechanism of transcriptional activation of human chorionic gonadotropin-alpha (hCGalpha) gene by epidermal growth factor (EGF) in trophoblast cells. We stably transfected hCGalpha promoter-chloramphenicol acetyltransferase constructs into Rcho-1 trophoblast cells and monitored the promoter activities. -290-base pair hCGalpha promoter containing a tandem repeat of cAMP response element (CRE) was activated by EGF in a dose- and time-dependent manner. Deletion analysis of hCGalpha promoter suggested an involvement of CRE in EGF-induced hCGalpha transcriptional activation. Moreover, the hCGalpha promoter, of which both CREs were mutated, did not respond to EGF. These results indicate that EGF activates the hCGalpha gene transcription through CRE. Although EGF did not alter the amount of CRE-binding protein (CREB), EGF induced CREB phosphorylation. We next examined the mechanism of CREB phosphorylation by EGF. Protein kinase C inhibitors (H7, staurosporin, and chelerythrine) inhibited EGF-induced CREB phosphorylation, whereas either mitogen-activated protein kinase kinase-1 inhibitor (PD98059) or protein kinase A inhibitor (H8) showed no effect. Furthermore, H7 and staurosporin but not H8 inhibited hCGalpha promoter activation by EGF. In conclusion, EGF promotes hCGalpha gene transcription via the CRE region probably by phosphorylating CREB mainly through the protein kinase C pathway in trophoblast cells.

Leukemia inhibitory factor (LIF) is a cytokine that plays an important role during mouse embryogenesis. We showed that adenovirus E1A represses the interleukin-6 signal transduction pathway that uses the same JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor as LIF. Here, we report that the LIF-JAK-STAT signal transduction pathway is blocked in cellular E1A-expressing undifferentiated F9 cells, and that the block is overcome by retinoic acid-induced differentiation. LIF failed to stimulate the expression of the acute phase response element (APRE)-driven luciferase gene in undifferentiated F9 cells, whereas the luciferase activity was remarkably increased by LIF treatment in differentiated F9 (dF9) cells. We analyzed the mechanism of the APRE regulation and found that the LIF-induced APRE-binding activity was regulated in a differentiation-dependent manner. The protein levels and the tyrosine phosphorylation of JAK1, JAK2, and STAT3 in F9 cells were not different from those in dF9 cells. The exogenous expression of activated c-Ha-ras partially recovered the LIF responsiveness of the APRE-luciferase gene in F9 cells, but the dominant negative ras N-17 did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. These results suggested that an unknown coactivation process that is partially compensated by Ras is required for STAT3-APRE binding in F9 cells.

The signal transduction mechanism involved in human placental lactogen (hPL) was studied. We have identified that hPL rapidly stimulated the tyrosine phosphorylation of at least 7 proteins including Janus Kinases (JAK1 and JAK2) and a signal transducer and activator of transcription protein (Stat3). This is the first evidence that the JAK-STAT pathway is involved in the hPL signaling. Moreover, two unknown proteins which were different from STAT proteins (Stat1, 3 and 5) in sizes were predominantly tyrosine-phosphorylated. Because human growth hormone (hGH) activates Stat1, 3, 5 and human prolactin (hPRL) activates Stat5, these results show that hPL uses a unique signal transduction pathway which is different from hGH and hPRL.

Background: The regulatory mechanism of tumor markers secretion has not been well clarified., Methods: Serum levels of CA 125 and tissue polypeptide antigen (TPA) from 17 patients with Stage III serous cystadenocarcinoma were measured prior to an initial surgical treatment. Epidermal growth factor receptor (EGFR) status was examined by an 125I-EGF binding assay in a human serous cystadenocarcinoma cell (SHIN-3) and in the 17 primary carcinomas. SHIN-3 cell and the EGFR-expressing primary cancer cells (n = 4) were cultured with or without various concentrations of transforming growth factor (TGF-alpha), a ligand for EGFR, and the CA 125 and TPA concentrations in the conditioned media were measured., Results: EGFR was expressed in 12 primary carcinomas and in the SHIN-3 cell, and it was absent in the remaining 5 carcinomas. Pre-therapeutic serum CA 125 and TPA levels were significantly greater (P < 0.05) in patients with EGFR-expressing carcinomas (n = 5). These data suggest a possible involvement of EGFR in regulating these tumor markers secretion. TGF-alpha increased the CA 125 and TPA secretion from SHIN-3 cell. It also promoted the CA 125 secretion in 2 of 4 EGFR-expressing primary ovarian carcinoma specimens., Conclusions: These results suggest that a signal through the EGFR may be involved in regulating the CA 125 and TPA secretion from human ovarian carcinomas.

We prepared a specific antibody against GIRK1, the major subunit of the G protein-gated K+ (KG) channel. Immunohistochemical study revealed that GIRK1 immunoreactivity was detected in the presynaptic, but not in the postsynaptic, region in the paraventricular nucleus of the rat hypothalamus (PVN). Therefore, activation of the KG channel may underlie presynaptic inhibition of neurotransmitter release by various agonists, such as dopamine, noradrenaline, opioids, and histamine, in PVN.

Distribution of IRK2 inwardly rectifying potassium channel mRNA in the mouse brain was studied using in situ hybridization histochemistry and compared with those of other classical inwardly rectifying potassium channel (IRK1 and IRK3) mRNAs. All these IRK channel mRNAs were detected in neurons, but not in glial cells. Their distribution patterns in the brain were, however, quite divergent: IRK2 mRNA was detected extremely high in granule cells of cerebellum, relatively high in motor trigeminal nucleus and moderate in olfactory bulb, piriform cortex, cerebral cortex, CA1 through CA3 regions of hippocampus, dentate gyrus and pontine nucleus. On the other hand, IRK1 mRNA was expressed throughout whole brain but in particular subsets of neurons, and IRK3 mRNA was in forebrain. Expression of these three IRK mRNAs overlapped in hippocampus, olfactory bulb, and cerebral cortex. This differential distribution of IRK mRNAs suggests that each of these channels has its specific function in regulation of the excitability of brain neurons.

beta gamma Subunits of heterotrimeric GTP-binding proteins (G beta gamma) activate the inwardly rectifying muscarinic K+ channel, GIRK1. The significant role for the carboxyl (C) terminus of GIRK1 in this interaction has been suggested. However, it is still unknown whether G beta gamma directly interacts with GIRK1. To elucidate the molecular basis of G beta gamma-activation of GIRK1, we examined the binding properties of G beta gamma to the C terminus of GIRK1 cloned from mouse brain cDNA library (MB-GIRK1). The C terminus of MB-GIRK1 fused with glutathione S-transferase directly bound to purified G beta gamma. Incubation of the C terminus with Gi pretreated with GTP gamma S, but not with GDP, resulted in the binding of Gi beta gamma to the protein. Purified G alpha-GDP, but not G alpha-GTP gamma S, inhibited the binding of G beta gamma to the fusion protein. These results indicate that G beta gamma dissociated from G alpha may directly bind to the C terminus of GIRK1.

We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.

The inhibition of carbocyclic inosine (C-Ino), 3'-deoxyinosine (3'-dI), and 3'-fluoroinosine (3'-FI) to Leishmania donovani amastigotes was examined. J774.1 cells (a mouse macrophage line) were cultured in GIT medium with lipopolysaccharide and hemin and infected with the parasite. C-Ino (3 microM) completely inhibited and 3'-dI (30 microM) reduced to 40% the infection rate on Day 6 after infection. Pentostam (30 microM) resulted in a 38% infection rate. The therapeutic efficacies of nonentrapped free and liposome-entrapped inosine analogs were tested in mice infected with L. donovani. The mice were injected intravenously five times on alternate days, beginning 2 days after infection. Treatment with the nonentrapped free inosine analog of C-Ino (100 mg/kg), 3'-dI (100 mg/kg), or 3'-FI (50 mg/kg) resulted in an LDU that was 94, 68, or 73% lower, respectively, than the control values. Treatment with the corresponding entrapped inosine analog (10 mg/kg) caused decreases of 90, 69, or 68% LDU, respectively. The entrapped inosine analogs were inhibitory at doses one-fifth to one-tenth of the nonentrapped free inosine analogs. C-Ino had the strongest inhibitory effect among the three analogs tested in vitro and in vivo. Liposome-entrapped C-Ino had no severe side effects, although spleen weight increased. The agent may be useful as an anti-leishmanial drug.

We studied the estrogen-dependent expression of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and EGF receptor gene transcripts in human fallopian tubes in vivo and in vitro. Competitive polymerase chain reaction (PCR) was performed on the fallopian tube RNA samples from the postmenopausal women with or without estrogen replacement. Amounts of EGF, TGF alpha, and EGF receptors gene transcripts in the estrogen-treated group (n = 3) were significantly (P < 0.01) more than those in the untreated group (n = 3). Competitive PCR also showed that EGF, TGF alpha, and EGF receptor gene transcripts level in tubal cells were increased by estrogen in vitro: messenger RNA levels of these factors were significantly (P < 0.01, n = 3) increased in cells incubated with 10(-8) M estrogen compared with those in cells without estrogen treatment. We studied whether EGF and/or TGF alpha is involved in the estrogen-induced tubal cell growth in vitro. Estrogen enhanced the [3H]-thymidine incorporation into the cell in dose- and time-dependent manners in culture: estrogen treatment for more than 12 h significantly (P < 0.05) enhanced the [3H]-thymidine incorporation into the cell at 10(-8) M. The estrogen-induced cell growth was observed in association with the increase in EGF, TGF alpha, and EGF receptor messenger RNA levels by estrogen. If the EGF and/or TGF alpha is involved in the cell growth, then the estrogen-induced cell growth should be suppressed by blocking the action of EGF and/or TGF alpha. Therefore, we examined the effects of neutralizing monoclonal antibodies against EGF, TGF alpha, and EGF receptors. Anti-EGF antibody significantly reduced the estrogen-induced increase in [3H]-thymidine incorporation, whereas anti-TGF alpha antibody failed to show the effect. Anti-EGF receptor antibody showed a significant suppressive effect on the estrogen-induced increase in [3H]-thymidine incorporation. Moreover, the growth inhibitory effect by 1 microgram/ml anti-EGF was restored by 10(-8) M EGF but not by TGF alpha even at 10(-6) M. All these data suggest that estrogen induces EGF and TGF alpha/EGF receptors in the human fallopian tube and that EGF but not TGF alpha may be involved in the estrogen-induced human tubal cell growth in vitro.

Background: The importance of epidermal growth factor (EGF) receptor-dependent growth has not been clarified for in vivo growth of primary human ovarian cancers., Methods: Seventeen primary human ovarian cancer tissue samples were examined for the presence of EGF receptors by a 125I-EGF-binding study. Three groups of mice were inoculated with EGF receptor expressing and not-expressing cancer tissues. The groups were as follows: control group, Sx group (mice that underwent sialoadenectomy; EGF depleted mice), and Sx+EGF (EGF-replaced) group. The ability of the inoculated tissues to implant and grow then was studied., Results: Of the 17 primary ovarian cancers, 12 expressed EGF receptors and 5 did not. Eight of 12 EGF-receptor expressing cancer tissues implanted and formed growing tumors in control animals. None implanted in the Sx animals. Epidermal growth factor receptor-expressing cancers implanted in Sx animals that received EGF administration. Two of five EGF receptor-negative ovarian cancers implanted and grew in both control and Sx animals., Conclusion: Growth of EGF receptor-expressing primary human ovarian cancers may be dependent on EGF in vivo.

We have reported the importance of epidermal growth factor (EGF) for the induction of obesity in mice. In this study, we studied the effects of EGF on the induction of lipogenic enzymes and on the accumulation of triglyceride in a differentiated mouse adipocyte cell in vitro. Mouse 3T3-L1 preadipocytic cells differentiated into mature adipocytes after the differentiation procedure by insulin, dexamethasone, and methyl-isobutyl-xanthine. 125I-EGF binding studies in the differentiated 3T3-L1 cells showed specific 125I-EGF bindings, and they expressed gene transcripts for EGF receptors by reverse transcription and polymerase chain reaction at all differentiative stages examined. Although EGF showed inhibitory effects on the triglyceride accumulation when administered to the preadipocytic 3T3-L1 cells, EGF enhanced the adipogenesis in the differentiated cells in dose- and time-dependent manners. Administration of EGF at 0.1-1 nM from 4 days after the differentiation procedure for 10 days, significantly enhanced the acyl-Co A synthetase and lipoprotein lipase messenger RNA levels, both of which are rate-limiting enzymes to synthesize triglyceride in adipocytes. Moreover, 0.1-1 nM EGF increased the amounts of triglyceride accumulated in the cells, in proportion to the acyl-Co A synthetase and lipoprotein lipase messenger RNA levels. EGF rather failed the adipogenesis at 10 nM. Time course studies revealed that 1 nM EGF significantly increased the intracellular triglyceride levels from 4 through 16 days administration. These results suggest that EGF shows biphasic effects on adipocytes: although EGF inhibits preadipocytes differentiation into mature adipocytes, it promotes adipogenesis in the differentiated adipocytes.