Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

Insulin is a critical signaling molecule in reducing blood glucose levels, and pyruvate dehydrogenase (PDH) is an essential enzyme in regulating glucose metabolism. However, the insulin effect on PDH function has not been well established. We observed that insulin attenuated the phosphorylation (p) of Ser264 (p-Ser264) in the PDH E1α subunit (PDHA1) in normal rat hepatocyte. In contrast, insulin induced an increase of p-Ser264 PDHA1 levels in hepatocellular carcinoma HepG2 and Huh7 cells. Insulin activated RhoA and Rho-dependent coiled coil kinase, an effector protein of active RhoA, which regulated p-Ser264 PDHA1 levels, along with both p-Ser9 and p-Tyr216 forms of glycogen synthase kinase-3ß (GSK-3ß) in HepG2 cells. Only p-Tyr216 GSK-3ß, the active form was involved in an increase of p-Ser264 PDHA1. Akt was also engaged in p-Ser9 of GSK-3ß, but neither in p-Tyr216 of GSK-3ß nor p-Ser264 of PDHA1 upon insulin. Reconstituted dephospho-mimic forms PDHA1 S264A and GSK-3ß Y216F impaired, but wild-types PDHA1 and GSK-3ß and phospho-mimic forms PDHA1 S264D and GSK-3ß Y216E increased cell proliferation upon insulin through expression of c-Myc and cyclin D1. Therefore, we propose that insulin-mediated p-PDHA1 is involved in the regulation of HepG2 cell proliferation through RhoA signaling pathway.-Islam, R., Kim, J.-G., Park, Y., Cho, J.-Y., Cap, K.-C., Kho, A.-R., Chung, W.-S., Suh, S.-W., Park, J.-B. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

The Experience of Human Milk Banking for 8 Years: Korean Perspective.

Human milk banks are a solution for mothers who cannot supply their own breast milk to their sick or hospitalized infants; premature infants, in particular, are unable to receive a full volume of breast milk for numerous reasons. As of December 2015, there was only one milk bank in a university hospital in Korea. We reviewed the basic characteristics of donors and recipients, and the amounts and contamination of breast milk donated at the Human Milk Bank in Kyung Hee University Hospital at Gangdong in Korea from 2008 to 2015. The donor pool consisted of 463 first-time donors and 452 repeat donors who made 1,724 donations. A total of 10,820 L of breast milk was collected, and 9,541.6 L were processed. Detectable bacteria grew in 12.6% after pasteurization and 52.5% had cytomegalovirus DNA before pasteurization in donated milk. There were 836 infant and 25 adult recipients; among new infant recipients, 48.5% were preterm; the groups received 8,009 and 165.7 L of donor milk, respectively. There was an increase in the percentage of preterm infants among new infant recipients in 2015 (93.1%) compared to 2008 (8.5%). Based on the number of premature infants in Korea, the number of potential recipients is not likely to diminish anytime soon, despite efforts to improve the breastfeeding rate. Sustainability and quality improvement of the milk bank need long-term financial support by health authorities and a nationwide network similar to blood banking will further contribute to the progress of milk banking.

Extracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition.

In the tumor microenvironment (TME), communication between cancer cells and tumor-associated macrophages (TAMs) through secreted extracellular proteins promotes cancer progression. Here, we observed that co-culturing cancer cells (4T1) and macrophage cells (Raw264.7) significantly enhanced superoxide production in both cell types. Using MALDI-TOF, we identified PKM2 as a highly secreted protein by Raw264.7 cells and bone marrow-derived monocytes. The extracellular recombinant PKM2 protein not only enhanced cancer cell migration and invasion but also increased superoxide production. Additionally, PKM2 was found to associate with the cell surface, and its binding to integrin α5/ß1 receptor was inhibited by antibodies specifically targeting it. Furthermore, we investigated downstream signaling pathways involved in PKM2-induced superoxide production. We found that knock-down of RhoA and p47phox using siRNAs effectively abolished superoxide generation in response to extracellular PKM2. Notably, extracellular PKM2 triggered the phosphorylation of p47phox at Ser345 residue and RhoA at Tyr42 residue (p-Tyr42 RhoA). Moreover, extracellular PKM2 exerted regulatory control over the expression of key epithelial-mesenchymal transition (EMT) markers, including ZEB1, Snail1, vimentin, and E-cadherin. Interestingly, p-Tyr42 RhoA translocated to the nucleus, where it bound to the ZEB1 promoter region. In light of these findings, we propose that extracellular PKM2 within the TME plays a critical role in tumorigenesis by promoting cancer cell migration and invasion through RhoA/p47phox signaling pathway.

Regulation of RhoA GTPase and novel target proteins for ROCK.

Rho GTPases play significant roles in cellular function and their activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), providing activation and inactivation of these GTPases, respectively. Active GTP-bound form of RhoA activates its effector proteins while the inactive GDP-bound form of RhoA exists in a RhoA-RhoGDI (guanine nucleotide dissociation inhibitor) complex in the cytosol. In particular, IκB kinase γ IKKγ/NF-κB essential modulator (NEMO) plays a role as a GDI displacement factor (GDF) for RhoA activation through binding to RhoA-RhoGDI complex. Meanwhile, prion protein inactivates RhoA despite RhoA/RhoGDI association. Novel target proteins for Rho-associated kinase (ROCK) such as glycogen synthase kinase (GSK)-3ß and IKKß are recently discovered. Here, we elaborate on a post-translationally modified version of RhoA, phosphorylated at Tyr42 and oxidized at Cys16/20. This form of RhoA dissociates from RhoA-RhoGDI complex and activates IKKß on IKKγ/NEMO, thus providing possibly a critical role for tumourigenesis.

Estrogen and hypoxia regulate estrogen receptor alpha in a synergistic manner.

Hypoxia activates and degrades estrogen receptor alpha (ERalpha) in human breast cancer cells, which may play an important role in the development and progression of breast cancer. In this study, the synergistic effects of estrogen (E(2)) and hypoxia on ERalpha-mediated transactivation and ERalpha degradation were investigated. ERalpha-mediated transcriptional activity was synergistically increased by E(2) and hypoxia, as determined by the transient expression of ERalpha and ER-responsive reporter plasmids in HEK 293 cells. Twenty hours of E(2) and hypoxia treatment synergistically induced degradation of ERalpha by 95% via a proteasome-dependent pathway in MCF-7 cells. These results provide evidence that hypoxia may stimulate yet unknown factor(s), which can further stimulate ERalpha signal transduction pathways.

Effects of inhibiting the proteasomal degradation of estrogen receptor alpha on estrogen receptor alpha activation under hypoxic conditions.

Hypoxia, which is intimately associated with the biology of breast carcinomas, modulates the level of estrogen receptor (ER) alpha expression and transactivation. We investigated the effect of blocking ER degradation on ERalpha-mediated transactivation under hypoxic conditions using the proteasome inhibitor MG132. Pretreatment with MG132 blocked hypoxia-induced degradation of ERalpha protein. Our data imply that ERalpha proteasomal inhibition is linked to receptor transactivation under hypoxia.

Clinical Characteristics and Prognostic Factors in Hemophiliacs with Intracranial Hemorrhage: A Single-Center, Retrospective Experience.

Intracranial hemorrhage (ICH) is the most serious bleeding event that occurs in patients with hemophilia; its estimated mortality rate is approximately 20 %, accounting for the largest number of deaths from bleeding. We conducted this single-center, retrospective study to examine the characteristics of and prognostic factors in patients with hemophilia. A comprehensive review of 12 cases of intracranial hemorrhage (ICH) among 10 patients. All 12 cases of ICH in the 10 patients were treated with clotting factor concentrates. Three patients had intracerebral hemorrhage that required neurosurgical intervention. After presenting with ICH, two pediatric patients developed antibodies to clotting factors. Two adult patients with intracerebral hemorrhage died, and the mortality rate was thus 20.0 % (2/10) in our clinical series. Prompt and intensive treatment with clotting factor concentrates may significantly lower the mortality rate among patients with hemophilia presenting with ICH. Our results showed a better prognosis in pediatric patients with intracerebral hemorrhage. Clinicians should pay special attention to the possible development of inhibitors after intensive treatment in pediatric patients. Further studies are needed to examine methods for administering clotting factor concentrates and to determine whether neurosurgical intervention is essential in each case.

Ginsenoside Rc and Re stimulate c-fos expression in MCF-7 human breast carcinoma cells.

We have found that ginsenoside Rc and Re induce c-fos in MCF-7 human breast carcinoma cells at both the mRNA and protein levels. However, neither ginsenoside activated the expression of reporter gene under the control of AP-1/TPA response elements. We have also examined the possibility that ginsenoside Rc and Re act by binding to intracellular steroid hormone receptors that act as transcriptional factors in the nucleus in inducing c-fos mRNA in MCF7 human breast carcinoma cells. However, ginsenoside Rc and Re did not bind to glucocorticoid, androgen, estrogen, or retinoic acid receptors as examined by the transcription activation of the luciferase reporter genes in CV-1 cells that were transiently transfected with the corresponding steroid hormone receptors and hormone responsive luciferase reporter plasmids. These data demonstrate that ginsenoside Rc and Re act via other transcription factors and not via estrogen receptor in c-Fos expression.

Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells.

Ginseng has been recommended to alleviate the menopausal symptoms, which indicates that components of ginseng very likely contain estrogenic activity. We have examined the possibility that a component of Panax ginseng, ginsenoside-Rb1, acts by binding to estrogen receptor. We have investigated the estrogenic activity of ginsenoside-Rb1 in a transient transfection system using estrogen-responsive luciferase plasmids in MCF-7 cells. Ginsenoside-Rb1 activated the transcription of the estrogen-responsive luciferase reporter gene in MCF-7 breast cancer cells at a concentration of 50 microM. Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of ginsenoside-Rb1 is estrogen receptor dependent. Next, we evaluated the ability of ginsenoside-Rb1 to induce the estrogen-responsive gene c-fos by semi-quantitative RT-PCR assays and Western analyses. Ginsenoside-Rb1 increased c-fos both at mRNA and protein levels. However, ginsenoside-Rb1 failed to activate the glucocorticoid receptor, the retinoic acid receptor, or the androgen receptor in CV-1 cells transiently transfected with the corresponding steroid hormone receptors and hormone responsive reporter plasmids. These data support our hypothesis that ginsenoside-Rb1 acts a weak phytoestrogen, presumably by binding and activating the estrogen receptor.

Extracts from Schizandra chinensis fruit activate estrogen receptors: a possible clue to its effects on nitric oxide-mediated vasorelaxation.

Schizandra chinensis fruit has long been used for the treatment of cardiovascular symptoms associated especially with menopausal symptoms in Korea. To provide a scientific rationale for such uses, we have investigated the vasorelaxant effects of Schizandra chinensis fruit on the vasomotor tone of the rat thoracic aorta in an organ bath. The crude extracts of Schizandra chinensis fruit (SC-Ex) elicited a transient relaxing response in the endothelium-intact rat aorta contracted with norepinephrine. This relaxant effect was abolished by removal of the endothelium, and also by pretreatment with nitric oxide synthase inhibitor. We then examined whether this vasodilatory effect occurs through estrogen receptor by reporter assays. SC-Ex activated the estrogen-responsive luciferase gene in COS cells transiently transfected with estrogen receptor and reporter plasmids. The activation was maintained in the butanol-soluble fraction and further increased in the successively fractionated C(18) cartridge-adsorbed fraction (SC-ADF). Reporter gene activation by SC-ADF was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the effect is estrogen receptor dependent. However, SC-ADF failed to activate the androgen receptor in COS cells transfected with the corresponding receptor and reporter plasmids. These data show that extracts of Schizandra chinensis fruit act as a weak phytoestrogen.