Yong-Sung Juhnn

Human papillomavirus 16 E5 up-regulates the expression of vascular endothelial growth factor through the activation of epidermal growth factor receptor, MEK/ ERK1,2 and PI3K/Akt

Abstract.The E5 oncoprotein of human papillomavirus (HPV) 16 plays an important role in early cervical carcinogenesis. Vascular endothelial growth factor (VEGF) plays a central role in switching on the angiogenic phenotype during early cervical carcinogenesis. However, the relationship between E5 and VEGF has not previously been examined. To clarify the regulatory role of E5 in VEGF expression, we transferred the E5 gene into various cell types. E5 increased VEGF expression. The addition of epidermal growth factor receptor (EGFR) inhibitor significantly suppressed VEGF expression, demonstrating that E5 stimulates VEGF expression through the activation of EGFR. E5-mediated EGFR activation was accompanied by phosphorylation of Akt and ERK1/2, which are also involved in VEGF expression. Furthermore, the mRNA stability of VEGF was not affected by E5, but VEGF promoter activity could be modulated by inhibitors of the EGFR, MEK-ERK1/2 and PI3K/Akt pathways in E5-expressing cells. Collectively, these novel results suggest that HPV 16 E5 increases VEGF expression by activating EGFR, MEK/ERK1/2 and PI3K/Akt.

Akt involvement in paclitaxel chemoresistance of human ovarian cancer cells

Abstract:  Paclitaxel (taxol) is extensively used for chemotherapy of various cancers including ovarian cancer. Although paclitaxel induces apoptosis in cancer cells, its exact mechanism of action still remains to be determined. Akt mediates survival signals which preserve various cancer cells from apoptosis pathway. Thus, Akt is considered an exciting target for therapeutics. Here, we demonstrated that inhibition of Akt increases the efficacy of the paclitaxel‐induced apoptosis in SKOV3 and PA‐1 human ovarian cancer cells. The sensitivity to paclitaxel of SKOV3 and PA‐1 cells was examined using the MTT assay. At a concentration of 30 nM, PA‐1 cells were more sensitive to paclitaxel than SKOV3 cells. Apoptosis was accompanied by the release of cytochrome c into the cytoplasm and cleavage of poly (ADP‐ribose) polymerase (PARP). To further elucidate the mechanism of apoptosis by paclitaxel, we compared the levels of phosphorylation of Akt between paclitaxel‐resistant SKOV3 cells and paclitaxel‐sensitive PA‐1 cells. The higher level of phosphorylated Akt was shown in SKOV3 cells than in PA‐1 cells. Interestingly, the treatment of paclitaxel decreased the amount of phosphorylated Akt in a time‐dependent manner in both cell lines. Furthermore, inhibition of Akt by specific phosphatidyinositol‐3‐kinase (PI3K)‐Akt inhibitors (Wortmannin, and LY294002) synergistically increased the efficacy of the paclitaxel‐induced apoptosis in both cell lines. These results suggest that the addition of the Akt inhibitor may increase the therapeutic efficacy of paclitaxel for patients with ovarian cancer.

Biphasic changes in the Ser-9 phosphorylation of glycogen synthase kinase-3β after electroconvulsive shock in the rat brain

BACKGROUND Glycogen synthase kinase-3beta (GSK-3beta) plays important roles in intracellular signaling pathways. Phosphorylation at Ser-9 reduces the activity of GSK-3beta, while phosphorylation at Tyr-216 enhances its activity. Mood stabilizing agents increase the phosphorylation of GSK-3beta at Ser-9, and hence inhibit its activity. This property has been considered to be related to the therapeutic action of these drugs. The effect of electroconvulsive shock (ECS), an effective mood stabilizing treatment, on the phosphorylation of GSK-3beta is not known yet. METHODS In this study, the effect of ECS on the phosphorylation of GSK-3beta was examined in the rat frontal cortex, hippocampus, and cerebellum by Western blot analysis using antibodies specific for Ser-9 or Tyr-216 phosphorylated GSK-3beta. RESULTS In all regions, the phosphorylation of GSK-3beta at Ser-9 was decreased immediately after ECS, but then increased above basal level within 10 min and maintained at an increased level for more than 30 min. Meanwhile, the phosphorylation at Tyr-216 of GSK-3beta did not show any significant changes after ECS. CONCLUSIONS These results showed that ECS could induce biphasic changes in the Ser-9 phosphorylation of GSK-3beta in the rat brain, suggesting some similarities, as well as differences, in the actions of mood stabilizers and ECS in the signal transduction mechanisms of the brain.

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium‐dependent protein phosphatase, in dephosphorylating Ser‐9 and activating glycogen synthase kinase‐3β (GSK‐3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK‐3β at Ser‐9 in SH‐SY5Y human neuroblastoma cells. The over‐expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser‐9, an increase in the activity of GSK‐3β, and an increase in the phosphorylation of tau. Km of calcineurin for a GSK‐3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK‐3β were co‐immunoprecipitated in neuron‐derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK‐3β. We conclude that in vitro, calcineurin can dephosphorylate GSK‐3β at Ser‐9 and form a stable complex with GSK‐3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK‐3βin vivo.

Human papillomavirus type 16 E5 protein inhibits hydrogen-peroxide-induced apoptosis by stimulating ubiquitin-proteasome-mediated degradation of Bax in human cervical cancer cells.

To investigate the mechanism by which the human papillomavirus (HPV) E5 protein contributes to the carcinogenesis of uterine cervical cancer, we studied the effect of HPV E5 on apoptosis of cervical cancer cells and its underlying mechanism. Expression of HPV16 E5 protein inhibited hydrogen peroxide-induced apoptosis in C-33A cervical cancer cells. E5 decreased the expression of Bax protein, and exogenous expression of Bax abolished the anti-apoptotic effect of E5. Knockdown of E5 by small interfering RNA sensitized CaSki cervical cancer cells to hydrogen peroxide-induced apoptosis with concurrent increase in Bax expression. Transient expression of E5 significantly increased the degradation rate of Bax protein by inducing the ubiquitination. The E5-induced decrease in Bax expression was inhibited by a cyclooxygenase-2 (COX-2) inhibitor, prostaglandin E2 (PGE(2)) receptor antagonists and cyclic adenosine monophosphate-dependent protein kinase (PKA) inhibitor. Treatment with PGE(2) decreased the expression of Bax and inhibited hydrogen peroxide-induced apoptosis of C-33A cells. We concluded that HPV16 E5 protein inhibits hydrogen peroxide-induced apoptosis of cervical cancer cells by stimulating the ubiquitin-proteasome-mediated degradation of Bax protein, and the pathway involves COX-2, PGE(2) and PKA. This finding suggests the possibility that HPV 16 E5 protein contributes to cervical carcinogenesis by inhibiting apoptosis of transformed cervical epithelial cells.

Human papillomavirus E5 protein induces expression of the EP4 subtype of prostaglandin E2 receptor in cyclic AMP response element-dependent pathways in cervical cancer cells

Human papillomavirus (HPV) is the major cause of uterine cervical cancer, but the role of the HPV E5 in carcinogenesis is not clearly understood. Prostaglandins are known to contribute to carcinogenesis of cervical cancer, and we therefore investigated the effect of HPV16 E5 on the expression of prostaglandin E2 (PGE2) receptors and underlying mechanisms. Stable expression of the E5 induced expression of the EP4 subtype of PGE2 receptors in C33A cervical cancer cells, and transfection of E5 small interfering RNA (siRNA) decreased it. EP4 protein expression was increased in human cervical cancer tissues, and EP4 mediated E5-induced increase in anchorage-independent colony formation and vascular endothelial growth factor expression. E5 induced cyclooxygenase-2 (COX-2) expression, and COX-2 increased PGE2 secretion and EP4 expression. The induction of EP4 by PGE2 and E5 was inhibited by an EP4 antagonist, inhibitors of cyclic adenosine monophosphate-dependent protein kinase or phosphatidylinositol 3-kinase, and a cyclic adenosine monophosphate response element (CRE) decoy. E5 increased the luciferase expression controlled by a variant CRE of the EP4 promoter, and it also increased the binding of cyclic adenosine monophosphate response element binding protein (CREB) to oligonucleotides containing this CRE. We conclude that the HPV16 E5 protein induces EP4 receptor protein in cervical cancer cells and that this induction involves epidermal growth factor receptor, COX-2, PGE2, EP2 and EP4, protein kinase A, CREB and CRE.

Suppression of the carbachol-activated nonselective cationic current by antibody against alpha subunit of Go protein in guinea-pig gastric myocytes

Abstract In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 µM carbachol (ICCh) at a holding potential of –60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as ICCh was slowly activated by the dialysis of guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block ICCh, antibodies (Abs) against Gα,i (anti-Gα,i) or Gα,o (anti-Gα,o) were tested. Activation of ICCh was blocked by the addition of anti-Gα,o. However, anti-Gα,i Abs had no significant effect on ICCh.The expression of Gα,o in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that Go-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes.

Differential changes in the expression of cyclic nucleotide phosphodiesterase isoforms in rat brains by chronic treatment with electroconvulsive shock

Electroconvulsive shock (ECS) has been suggested to affect cAMP signaling pathways to exert therapeutic effects. ECS was recently reported to increase the expression of PDE4 isoforms in rat brain, however, these studies were limited to PDE4 family in the cerebral cortex and hippocampus. Thus, for comprehensive understanding of how ECS regulates PDE activity, the present study was performed to determine whether chronic ECS treatment induces differential changes in the expression of all the PDE isoforms in rat brains. We analyzed the mRNA expression of PDE isoforms in the rat hippocampus and striatum using reverse transcription polymerase chain reaction. We found chronic ECS treatment induced differential changes in the expression of PDE isoform 1, 2, 3, 4, 5 and 7 at the rat hippocampus and striatum. In the hippocampus, the expression of PDE1A/B (694%), PDE4A (158%), PDE4B (323 %), and PDE4D (181%) isoforms was increased from the controls, but the expression of PDE2 (62.8%) and PDE7 (37.8%) decreased by chronic ECS treatment. In the striatum, the expression of PDE1A/B (179%), PDE4A (223%), PDE4B (171%), and PDE4D (327%) was increased by chronic ECS treatment with the concomitant decrease in the expression of PDE2 (78.4%) and PDE3A (67.1%). In conclusion, chronic ECS treatment induces differential changes in the expression of most PDE isoforms including PDE1, PDE2, PDE3, PDE4, PDE5, and PDE7 in the rat hippocampus and striatum in an isoform- and brain region-specific manner. Such differential change is suggested to play an important role in regulation of the activity of PDE and cAMP system by ECS.

Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells

Peripheral primitive neuroectodermal tumour (PNET)/Ewings sarcoma (ES) and neuroblastoma (NB) are related tumours of neural crest origin with primitive neural characteristics. Fibroblast growth factor 2 (FGF2) is a critical signalling molecule for primitive neural crest cells. The treatment of NB cells with FGF2 variably affects biological characteristics such as growth and differentiation, while in PNET/ES, FGF2 predominantly induces apoptosis. The JK‐GMS Askin tumour cell line can be induced to differentiate upon treatment with nerve growth factor (NGF), indicating the integrity of the cellular machinery necessary for differentiation. The present study assesses whether FGF2 can induce differentiation in JK‐GMS cells. JK‐GMS cells expressed high‐affinity FGF receptors (FGFRs), and treatment with FGF2 induced phosphorylation of FGFR1 together with activation of extracellular signal‐regulated kinases (ERK1/ERK2) and c‐Jun N‐terminal kinase (JNK). Subsequent biological effects were growth inhibition, neuronal differentiation, and apoptosis, and these changes were associated with increased expression of neurofilaments, reduction of c‐myc and bcl‐2 expression, and activation of caspase 3. Treatment of the cells with a specific inhibitor of the MAPK/extracellular signal‐regulated kinase (MEK)‐1, PD98059, predominantly inhibited the effects of FGF2 on growth, differentiation, and apoptosis, while an inhibitor of JNK reduced apoptosis, indicating that the ERK1/2 and JNK pathways are critical components of FGF2‐mediated effects in JK‐GMS cells. Additional comparative analyses of FGF2‐mediated effects in two ES cell lines (CADO‐ES, RD‐ES) and a PNET cell line (SK‐N‐MC) showed pronounced differentiation in SK‐N‐MC, but not in CADO‐ES or RD‐ES cells. This study demonstrates that FGF2 can induce neuronal differentiation of PNET including Askin tumour. These findings clearly indicate that the FGF2‐mediated signalling pathway plays a critical role in controlling the major properties of PNET cells and may provide a potential therapeutic target for PNET. Copyright

Increased phosphorylation of Ser473-Akt, Ser9-GSK-3β and Ser133-CREB in the rat frontal cortex after MK-801 intraperitoneal injection

GSK-3beta is regarded as playing an important part in the pathogenesis of schizophrenia and the action of psychotomimetic agents. We observed phosphorylation of molecules associated with the GSK-3beta signalling pathway in the rat brain after MK-801 injection, which induces a schizophrenia-like state in humans. Ser9-GSK-3beta phosphorylation was increased after injection of 1 mg/kg MK-801 in the rat frontal cortex but not in the hippocampus or cerebellum. This increase peaked at 30 min and was maintained until 90 min after injection. The phosphorylation showed a dose-dependent increase up to 1 mg/kg MK-801, followed by a decrease at higher dosage. Furthermore, phosphorylation of Ser473-Akt and Ser133-CREB showed similar temporal, dose-dependent and regionally specific patterns with those of Ser9-GSK-3beta. However, phosphorylation of Dvl and Ser33-beta-catenin was not affected by MK-801. These results suggest that GSK-3beta phosphorylation by MK-801 may be associated with the Akt-GSK-3beta pathway rather than with the Wnt-Dvl-GSK3beta pathway.