Ligand-independent activation of steroid receptors

In several transformed cell lines, the growth factors IGF-I and epidermal growth factor (EGF) activate second messenger systems that cause the phosphorylation of the estrogen receptor (ER). One kinase catalysing receptor phosphorylation is mitogen activated protein (MAP) kinase, and the result of phosphorylation is an increase in receptor transactivation function. EGF and IGF-I, secreted locally and systemically, are involved in uterine-conceptus interactions in early pregnancy, and therefore it is of interest to determine whether these growth factors affect ER function in the uterus. An estrogen response element, chloramphenicol acetyl transferase reporter gene construct (CATERE) was transfected into bovine endometrial epithelial and stromal cells in vitro, and CAT measured during transient expression. Growth factors were added at various times following transfection, and MAP kinase phosphorylation was monitored by western blotting of p42 and p44. The MEK inhibitor U 0126 was used to determine whether the effect of IGF-I on CATERE expression was mediated through MAP kinase, and the anti-estrogen ICI 182780 was used to identify effects involving the ER. In stromal cells, reporter gene activity was increased in a dose dependent manner by IGF-I or hEGF in the presence or absence of estradiol-17beta. In the absence of estradiol the effect of IGF-I was not inhibited by ICI 182780. The effect of IGF-I occurred within an hour, before any detectable increase in cell proliferation, and the activation of CAT expression in response to IGF-I or EGF was blocked by U 0126. In contrast to their effects in stromal cells, neither IGF-I nor EGF affected CAT expression in bovine endometrial epithelial cells. Measurement of phosphorylated MAP kinases p42/p44 by western blotting showed that EGF but not IGF-I activated MAP kinase phosphorylation in both epithelial and stromal cells. In stromal cells, the fact that U 0126 blocked the CAT responses to IGF-I and EGF indicates the involvement of a MAP kinase. But since IGF-I did not activate p42/p44, a different MAP kinase, not detected by the antibody used here, is implicated. As the response was not blocked by ICI 182780, we conclude this effect is independent of ER activation. Therefore in bovine uterine cells in culture effects on MAP kinases p42/p44 can be dissociated from those on ERE-dependent gene expression, and reporter gene expression may be independent of ER activation.     

IGF-I retards proper development of acinar structures formed by bovine mammary epithelial cells via sustained activation of Akt kinase

Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.     

Possible involvement of phosphatidylinositol 3-kinase in the maintenance of metaphase II attest in porcine oocytes matured in vitro

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34^cdc2 kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34^cdc2 kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 µmol/L LY294002 did not affect either the MAPK or p34^cdc2 kinase activities, 50 µmol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Therefore the effect of 10 µmol/L Ca²⁺ ionophore which was reported as inducing a transient decrease of p34^cdc2 kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.     

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.     

Steroid receptor expression and HER-2/neu (c-erbB-2) oncoprotein in the uterus of cats with cystic endometrial hyperplasia-pyometra complex

The presence of oestrogen-alpha receptor (ER), progesterone receptor (PR), and HER-2/neu (c-erbB-2) oncoprotein in the uterine walls of 10 healthy cats and 20 subjects with cystic endometrial hyperplasia-pyometra (CEH-P) were evaluated. Lesions were graded according to the severity of cystic dilation, hyperplasia and inflammation, and were classified as normal, mild uterine hyperplasia and severe uterine hyperplasia. The ER, PR and c-erbB-2 expression in the endometrium, glandular epithelium, stromal fibroblasts and myometrial smooth muscle cells was quantified by immunohistochemistry. The ER, PR and c-erbB-2 staining patterns differed between normal uteri and uteri with CEH-P. The ER expression was tended to be higher in the endometrial surface and glandular epithelium in the severe hyperplasia group (P > 0.05) and significantly lower in the mild hyperplasia cases compared with normal endometrium (P < 0.05), whereas the PR expression in both severe and mild hyperplasia cases tended to be higher in stromal cells and glandular epithelium than those in the normal uteri. C-erbB-2 immunoreactivity was observed only in the endometrial surface and glandular epithelium of the uterine wall and immunostaining was found to be highest in cases with severe hyperplasia. As a conclusion, we suggest that c-erbB-2 oncoprotein may play a role in the pathogenesis of the CEH together with the ER and PR in cats, and that ER does not have a role in the mechanism of pyometra, whereas PR plays a role in the pathogenesis of both CEH and pyometra.     

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17beta- and trenbolone acetate-stimulated proliferation of cultured bovine satellite cells

Although numerous studies have shown that both androgenic and estrogenic steroids increase rate and efficiency of muscle growth in steers, there is little consensus as to their mechanism of action. A combined estradiol 17beta (E2)/trenbolone acetate (TBA) implant causes a significant increase in muscle IGF-I mRNA and both E2 and TBA stimulate a significant increase in IGF-I mRNA level in bovine satellite cell (BSC) cultures in media containing 10% fetal bovine serum (FBS). Consequently, increased IGF-I expression may play a role in anabolic-steroid-enhanced muscle growth. However, even though treatment of cultured BSC with E2 or TBA in media containing 1% IGFBP-3-free swine serum (SS) results in increased proliferation there is no effect on IGF-I mRNA expression, suggesting that increased IGF-I expression may not be responsible for anabolic-steroid-enhanced BSC proliferation. To further examine the role of estrogen, androgen and IGF-I receptors and their respective ligands in E2- and TBA-stimulated BSC proliferation, we assessed the effects of specific inhibitors on E2- or TBA-stimulated proliferation of BSC. Both ICI 182 780 (an estrogen receptor blocker) and flutamide (an inhibitor of androgen receptor) suppressed (p<0.05) E2- and TBA-stimulated BSC proliferation, respectively. JB1 (a competitive inhibitor of IGF-I binding to type I IGF receptor) reduced (p<0.05) both E2- and TBA-stimulated proliferation in BSC cultures. Both the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways play significant roles in the actions of IGF-I on proliferation and differentiation of myogenic cells. PD98059, an inhibitor of the MAPK pathway, and wortmannin, an inhibitor of the PI3K pathway, both suppressed (p<0.05) E2- and TBA-stimulated proliferation of cultured BSC. Our data suggest that IGF-I plays a role in E2- and TBA-stimulated proliferation of cultured BSC even in the absence of increased IGF-I expression.     

The effect of interferon-tau and ovarian steroids on the proliferation of bovine endometrial cells in vitro

The goal of this study was to evaluate the effect of various concentrations of interferon-tau (IFN-tau) with or without steroid hormones, 171 estradiol or progesterone, on the proliferation of bovine endometrial cells in vitro. Endometrial epithelial and stromal cells were isolated from the uterus of cows during the early estrus cycle (2-3 days) and incubated with different doses of IFN-tau with or without steroid hormones. The proliferation was determined by the MTT test in 48, 96, and 144 h of incubation. An antiproliferative activity of IFN-tau was observed both in epithelial and stromal cells cultured in RPMI 1640 medium supplemented with 10% FBS or. serum replacement. However, epithelial cells were more sensitive to antiproliferative action of interferon-tau. It;s activity was dose-and time-dependent. The inhibition of epithelial cell proliferation by 50% (ED50) was achieved at concentrations of 500 U/ml, 340 U/ml, and 8.8 U/ml of IFN-tau after 48, 96, and 144 h of incubation, respectively. None of the doses of IFN-tau (10-10.000 U/ml) used inhibited stromal cell proliferation in 50%. The most effective dose of IFN-tau inhibiting stromal cell proliferation was 10.000 U/ml, which decreased cell growth by 17.08%, 22.87%, and 2.6% after 48, 96, and 144 h of incubation, respectively. Steroid hormones, 17beta estradiol and progesterone, added to the culture of stromal cells with or without IFN-tau did not significantly modulate stromal cell growth. In contrast, a high concentration of progesterone (10(-5) M) alone significantly enhanced stromal cell growth. Progesterone at low, physiological concentrations (10(-7)- 10(-9) M) ameliorated the antiproliferative activity of IFN-tau, especially at the 10(-9 )M concentration. At this concentration, the stimulatory effect on stromal cell growth was observed. The mechanisms of such response are not entirely clear but may arise from the influence of IFN-tau on progesterone down regulation of its own receptor. Depicted activity of IFN-tau may find usefulness in therapy of neoplastic disorders.     

Is Mechanically Induced Cystic Endometrial Hyperplasia (CEH) a Suitable Model for Study of Spontaneously Occurring CEH in the Uterus of the Bitch?

Cystic endometrial hyperplasia (CEH) was experimentally reproduced by the intraluminal insertion of a silk wire in the uterus of metestral bitches to obtain a model for the study of spontaneously occurring CEH in the bitch. This mechanically reproduced CEH corresponded histologically very well to spontaneously occurring CEH. With respect to sex hormone receptor expressions, however, there was no similarity. In the mechanically induced CEH, progesterone receptor expression was reduced in the epithelial cells (surface epithelium and endometrial glands) and slightly increased in the stromal fibroblasts and myometrium as compared with the normal metestrus uterus. The oestrogen receptor expression in mechanically induced CEH was reduced in the epithelial cells (surface epithelium and endometrial glands) and more or less unchanged in the stromal fibroblast and myometrium as compared with the normal metestrus uterus. This is in contrast to the increased sex hormone receptor expressions in all uterine cell types, observed in spontaneously occurring CEH. The mechanically induced CEH model corresponds immunohistochemically more to early placentation and to pyometra in the bitch. Thus this experimental CEH model is not suitable for pathogenic studies of spontaneously occurring CEH in the bitch.     

Effects of nitric oxide and tumor necrosis factor-alpha on production of prostaglandin F2alpha and E2 in bovine endometrial cells

The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)alpha influence on the bovine endometrium. TNFalpha influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFalpha might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFalpha- and NO-regulated prostaglandin (PG) F(2alpha) and PGE(2) synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 microM) for 24 h. S-NAP strongly stimulated PGE(2) production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF(2alpha) production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFalpha and NO action, stromal cells were incubated for 24 h with TNFalpha or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 microM) or an inhibitor of phosphodiesterase (IBMX; 10 microM). When the cells were exposed to TNFalpha in combination with NOS inhibitor (L-NAME), TNFalpha-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFalpha on PGs production (P<0.05). The overall results suggest that TNFalpha augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE(2) production in epithelial cells.     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.     

Uterine diseases in cattle after parturition

Bacterial contamination of the uterine lumen is common in cattle after parturition, often leading to infection and uterine disease. Clinical disease can be diagnosed and scored by examination of the vaginal mucus, which reflects the presence of pathogenic bacteria such as Escherichia coli and Arcanobacterium pyogenes. Viruses may also cause uterine disease and bovine herpesvirus 4 (BoHV-4) is tropic for endometrial cells, causing a rapid cytopathic effect. The elimination of pathogens by the innate immune system is dependent on pattern recognition receptors binding pathogen-associated molecules. Uterine epithelial and stromal cells express receptors such as Toll-like Receptor 4 that binds E. coli lipopolysaccharide. The infertility associated with uterine disease is caused by damage to the endometrium and disruption of ovarian cyclic activity. Bacteria modulate endometrial prostaglandin secretion, and perturb ovarian follicle growth and function. Understanding the molecular basis of uterine disease will lead to novel approaches to treating infertility.     

Expression of Glucocorticoid Receptor α and Its Regulation in the Bovine Endometrium: Possible Role in Cyclic Prostaglandin F2α Production

Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8–12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17β (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

Masitinib as a chemosensitizer of canine tumor cell lines: a proof of concept study

Masitinib, a selective tyrosine kinase inhibitor, has previously been shown to enhance the antiproliferative effects of gemcitabine in human pancreatic cancer, demonstrating potential as a chemosensitizer. This exploratory study investigated the ability of masitinib to sensitize various canine cancer cell lines to doxorubicin, vinblastine, and gemcitabine. Masitinib strongly sensitized histiocytic sarcoma cells to vinblastine (>70-fold reduction in IC(50) at 5 μM masitinib), as well as osteosarcoma and mammary carcinoma cells to gemcitabine (>70-fold reduction at 5-10 μM). In addition, several cell lines were sensitized to doxorubicin (2-10-fold reduction at 10 μM). These data establish proof-of-concept that masitinib in combination with chemotherapeutic agents can generate synergistic growth inhibition in various canine cancers, possibly through chemosensitization. The findings justify further investigation into those combinations that may potentially yield therapeutic benefit.     

Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92-98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [125I]IGF-I was in the order of IGF-I greater than IGF-II greater than or equal to insulin. Although the [125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 X 10(-9) M, 7.5 X 10(-8) M, and 8.7 X 10(-8) M for satellite cells and 3.1 X 10(-9) M, 7.5 X 10(-8) M, and 9.6 X 10(-8) M for myotubes, respectively. Receptor cross-linking analysis using disuccinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [125I]IGF-I in the presence or absence of 1 X 10(-7) M IGF-I, IGF-II, or insulin. Receptor subunit species of Mr 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a Mr greater than 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.     

Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt

Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.     

Contribution of MX dynamin, oligoadenylate synthetase, and protein kinase R to anti-paramyxovirus activity of type 1 interferons in vitro

OBJECTIVE: To determine the contribution of MX dynamin, oligoadenylate synthetase (OAS), and double-stranded RNA-dependent protein kinase R (PKR) to the antiviral effects of type 1 interferons (IFNs) against bovine parainfluenza-3 virus (PI-3V) infection of Vero cells. SAMPLE POPULATION: Vero cell cultures. PROCEDURES: PI-3V yield was first compared between control and transfected type 1 IFNs-incompetent Vero cells expressing recombinant OAS or MX proteins. Afterwards, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) was used to scale the degree of PKR activation upon infection of Vero cells by PI-3V. RESULTS: Overexpression of OAS did not result in significantly decreased viral replication. Phosphorylated eIF2alpha forms, the hallmark of PKR activation, were not increased in IFNalpha-primed infected Vero cells. Although human MXA contributed to partial blockade of replication of bovine PI-3V, the antiviral effect was not as strong as that of IFNalpha. CONCLUSIONS AND CLINICAL RELEVANCE: The powerful anti-Paramyxovirus activity of type 1 IFNs is mediated by noncanonic pathways.     

表皮生长因子调控猪肠上皮细胞中钠依赖Ⅱb型磷转运蛋白表达的细胞信号通路研究

本研究旨在探讨表皮生长因子(EGF)调控猪小肠上皮细胞IPEC-J2中钠依赖Ⅱb型磷转运蛋白(NaPi-Ⅱb)表达的分子机制。试验分别用EGF受体酪氨酸激酶抑制剂(tyrphostin AG1478)、蛋白激酶A(PKA)抑制剂(H89)、蛋白激酶C(PKC)抑制剂(k4393)、p38抑制剂(SB203580)、细胞外信号调节激酶(ERK)抑制剂(PD98059)、c-Jun氨基末端激酶(JNK)抑制剂(anisomycin)与EGF共同处理IPEC-J2细胞,利用Western blot检测相关通路蛋白及目的蛋白(NaPi-Ⅱb)的表达水平。结果显示:相较于对照组,EGF处理后NaPi-Ⅱb表达水平显著降低(P0.05);相较于无抑制剂组,EGF受体、PKA、PKC、丝裂原活化蛋白激酶(MAPK)/p38、MAPK/ERK1/2、MAPK/JNK的特异性抑制剂处理IPEC-J2后,NaPi-Ⅱb表达水平显著提高(P0.05),其中添加MAPK/ERK1/2特异性抑制剂显著降低了MAPK/ERK1/2在Tyr204位点的磷酸化水平(P0.05),添加MAPK/JNK的特异性抑制剂显著降低了MAPK/JNK1/2/3在Thr183和Tyr185位点的磷酸化水平(P0.05),说明该2组抑制剂对该通路的抑制作用是通过降低上述位点的磷酸化水平实现的。本研究结果表明EGF受体、PKA、PKC、p38、ERK和JNK均介导了EGF调控IPEC-J2细胞中NaPi-Ⅱb的表达。     

Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells

ABSTRACT: Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.     

miR‐182 selectively targets HOXA10 in goat endometrial epithelium cells in vitro

Proper HOXA10 expression was essential for endometrial receptivity what was crucial for successful embryo implantation in mammalian. This study confirmed that miR‐182 regulated the expression levels of HOXA10 by binding to its 3′ UTR, selectively downregulated HOXA10 in goat endometrial epithelium cells (gEECs) but not stromal cell (gESCs) in vitro. However, HOXA10 and miR‐182 both up‐expressed in the goat endometrium at gestational day 15 (D15) compared with gestational day 5 (D5), suggesting that there were some other factors regulated the expression of HOXA10 during the development of goat endometrium in vivo. What's more, HOXA10 gene silencing (HOXA10‐siRNA) resulted in gEECs apoptosis in vitro, and it regulated the protein levels of oestrogen receptor a (ERa), progesterone receptor B (PRb), insulin‐like growth factor 1 receptor (IGF1R), BCL‐2, pleiotrophin (PTN), AKT and p‐JNK in gEECs. Furthermore, HOXA10 might regulate the protein levels of endometrial receptivity biomarker genes, including vascular endothelial growth factor (VEGF), osteopontin (OPN), cyclooxygenase‐2 (COX‐2) and prolactin receptor (PRLR) in gEECs. In conclusion, miR‐182 targeted HOXA10 selectively in EECs in vitro, and HOXA10 played an important role in maintaining the function of EECs in dairy goats.