Benxu Cheng

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte-Like MLO-Y4 Cells†

Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte‐like cell line MLO‐Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO‐Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO‐Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO‐Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. “Scrape‐loading” dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte‐like cells was not observed in osteoblast‐like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte‐like MLO‐Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

PGE2 Is Essential for Gap Junction-Mediated Intercellular Communication between Osteocyte-Like MLO-Y4 Cells in Response to Mechanical Strain

We have observed, in our previous studies, that fluid flow increases gap junction-mediated intercellular coupling and the expression of a gap junction protein, connexin 43, in osteocyte-like MLO-Y4 cells. Interestingly, this stimulation is further enhanced during the poststress period, indicating that a released factor(s) is likely to be involved. Here, we report that the conditioned medium obtained from the fluid flow-treated MLO-Y4 cells increased the number of functional gap junctions and connexin 43 protein. These changes are similar to those observed in MLO-Y4 cells directly exposed to fluid flow. Fluid flow was found to induce PGE(2) release and increase cyclooxygenase 2 expression. Treatment of the cells with PGE(2) had the same effect as fluid flow, suggesting that PGE(2) could be responsible for these autocrine effects. When PGE(2) was depleted from the fluid flow-conditioned medium, the stimulatory effect on gap junctions was partially, but significantly, decreased. Addition of the cyclooxygenase inhibitor, indomethacin, partially blocked the stimulatory effects of mechanical strain on gap junctions. Taken together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated, in part, by the release of PGE(2). Hence, PGE(2) is an essential mediator between mechanical strain and gap junctions in osteocyte-like cells.

Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factors) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin E2 (PGE2) release and increase cyclooxygenase 2 (COX-2) expression. When PGE2 was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE2. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.

Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

Inhibition of proteasome activity and the resulting protein accumulation are now known to be important events in the development of many neurological disorders, including Alzheimers and Parkinsons diseases. Abnormal or over expressed proteins cause endoplasmic reticulum and oxidative stress leading to cell death, thus, normal proteasome function is critical for their removal. We have shown previously, with cultured SH-SY5Y neuroblastoma cells, that proteasome inhibition by the drug epoxomicin results in accumulation of ubiquitinated proteins. This causes obligatory loading of the mitochondria with calcium (Ca(2+)), resulting in mitochondrial damage and cytochrome c release, followed by programmed cell death (PCD). In the present study, we demonstrate that all-trans-retinoic acid (RA) pretreatment of SH-SY5Y cells protects them from PCD death after subsequent epoxomicin treatment which causes proteasome inhibition. Even though ubiquitinated protein aggregates are present, there is no evidence to suggest that autophagy is involved. We conclude that protection by RA is likely by mechanisms that interfere with cell stress-PCD pathway that otherwise would result from protein accumulation after proteasome inhibition. In addition, although RA activates both the AKT and ERK phosphorylation signaling pathways, only pretreatment with LY294002, an inhibitor of PI3-kinase in the AKT pathway, removed the protective effect of RA from the cells. This finding implies that RA activation of the AKT signaling cascade takes precedence over its activation of ERK1/2 phosphorylation, and that this selective effect of RA is key to its protection of epoxomicin-treated cells. Taken together, these findings suggest that RA treatment of cultured neuroblastoma cells sets up conditions under which proteasome inhibition, and the resultant accumulation of ubiquitinated proteins, loses its ability to kill the cells and may likely play a therapeutic role in neurodegenerative diseases.

Insulin-like growth factor-I mediates neuroprotection in proteasome inhibition-induced cytotoxicity in SH-SY5Y cells.

The proteasome is an enzyme complex responsible for targeted intracellular proteolysis. Alterations in proteasome-mediated protein clearance have been implicated in the pathogenesis of aging, Alzheimers disease (AD) and Parkinsons disease (PD). In such diseases, proteasome inhibition may contribute to formation of abnormal protein aggregates, which in turn activate intracellular unfolded protein responses that cause oxidative stress and apoptosis. In this study, we investigated the protective effect of Insulin-like Growth Factor-I (IGF-1) for neural SH-SY5Y cells treated with the proteasomal inhibitor, Epoxomicin. In SH-SY5Y cells, Epoxomicin treatment results in accumulation of intracellular ubiquitinated proteins and cytochrome c release from damaged mitochondria, leading to cell death, in Epoxomicin time- and dose-dependent manner. In cells treated with small amounts of IGF-1, the same dosages of Epoxomicin reduced both mitochondrial damage (cytochrome c release) and reduced caspase-3 activation and PARP cleavage, both of which are markers of apoptosis. Notably, however, IGF-1-treated SH-SY5Y cells still contained ubiquitinated protein aggregates. This result indicates that IGF-1 blocks the downstream apoptotic consequences of Epoxomicin treatment leading to decreased proteasome function. Clues as to the mechanism for this protective effect come from (a) increased AKT phosphorylation observed in IGF-1-protected cells, vs. cells exposed to Epoxomicin without IGF-1, and (b) reduction of IGF-1 protection by pretreatment of the cells with LY294002 (an inhibitor of PI3-kinase). Together these findings suggest that activation of PI3/AKT pathways by IGF-1 is involved in IGF-1 neuroprotection against apoptosis following proteasome inhibition.

G Protein-Coupled Estrogen Receptor Is Apoptotic and Correlates with Increased Distant Disease-Free Survival of Estrogen Receptor-Positive Breast Cancer Patients

Purpose: G protein–coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells. Experimental Design: GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R). Results: GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells. Conclusions: GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells. Clin Cancer Res; 19(7); 1681–92. ©2013 AACR.

N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells

Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinsons disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration.

Morphological diversity of cultured cold-active lytic bacteriophages isolated from the Napahai plateau wetland in China.

In summary, rod-shaped bacterial cells and Pseudomonas spp. dominated the bacterial community in the Napahai plateau wetland. Based on our TEM results, bacteriophages with larger capsids (60–140 nm) and Siphoviridae members were dominant. We were able to isolate a batch of cultured cold-active bacteriophages and their host bacterial strains, which should provide opportunities for further qualitative or quantitative analysis of bacteriophage function in wetland ecology. Specifically, these new isolates can help clarify bacteriophage coldadaptation mechanisms, structure, and assembly, as well as improve our understanding of phage-host co-evolution and interactions.

Niclosamide induces protein ubiquitination and inhibits multiple pro-survival signaling pathways in the human glioblastoma U-87 MG cell line

Glioblastoma is the most common and lethal malignant primary brain tumor for which the development of efficacious chemotherapeutic agents remains an urgent need. The anti-helminthic drug niclosamide, which has long been in use to treat tapeworm infections, has recently attracted renewed interest due to its apparent anticancer effects in a variety of in vitro and in vivo cancer models. However, the mechanism(s) of action remains to be elucidated. In the present study, we found that niclosamide induced cell toxicity in human glioblastoma cells corresponding with increased protein ubiquitination, ER stress and autophagy. In addition, niclosamide treatment led to down-regulation of Wnt/β-catenin, PI3K/AKT, MAPK/ERK, and STAT3 pro-survival signal transduction pathways to further reduce U-87 MG cell viability. Taken together, these results provide new insights into the glioblastoma suppressive capabilities of niclosamide, showing that niclosamide can target multiple major cell signaling pathways simultaneously to effectively promote cell death in U-87 MG cells. Niclosamide constitutes a new prospect for a therapeutic treatment against human glioblastoma.