Different susceptibilities of osteoclasts and osteoblasts to glucocorticoid-induced oxidative stress and mitochondrial alterations.

Glucocorticoid-induced bone loss is the most common form of secondary osteoporosis. This toxic effect has not been efficiently managed, possibly due to the incomplete understanding of the extraordinarily diverse cellular responses induced by glucocorticoid treatment. Previous literatures revealed that high dose of exogenous glucocorticoid triggers apoptosis in osteocytes and osteoblasts. This cell death is associated with glucocorticoid-induced oxidative stress. In this study, we aimed to investigate the mechanisms of glucocorticoid-induced apoptosis in osteoblasts and examine the responses of osteoclasts to the synthetic glucocorticoid, dexamethasone. We demonstrated the biphasic effects of exogenous glucocorticoid on osteoblastic mitochondrial functions and elevated intracellular oxidative stress in a dose- and time-dependent manner. On comparison, similar treatment did not induce mitochondrial dysfunctions and oxidative stress in osteoclasts. The production of reactive oxygen/nitrogen species was decreased in osteoclasts. The differences are not due to varying efficiency of cellular antioxidant system. The opposite effects on nitrogen oxide synthase might provide an explanation, as the expression levels of nos2 gene are suppressed in the osteoclast but elevated in the osteoblast. We further revealed that glucocorticoids have a substantial impact on the osteoblastic mitochondria. Basal respiration rate and ATP production were increased upon 24 h incubation of glucocorticoids. The increase in proton leak and nonmitochondrial respiration suggests a potential source of glucocorticoid-induced oxidative stress. Long-term incubation of glucocorticoids accumulates these detrimental changes and results in cytochrome C release and mitochondrial breakdown, consequently leading to apoptosis in osteoblasts. The mitochondrial alterations might be other sources of glucocorticoid-induced oxidative stress in osteoblasts.

Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy.

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity.

Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species - Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita - were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 (mu)g/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40-100 (mu)g/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.

Site-specific N-glycosylation of caprine lysostaphin restricts its bacteriolytic activity toward Staphylococcus aureus.

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goat ß-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn(125) but not Asn(232) is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn(232) to Gln(232) substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn(125) to Gln(125) substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn(125) of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.

Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells.

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

Protective effects of adiponectin against renal ischemia-reperfusion injury via prostacyclin-PPARα-heme oxygenase-1 signaling pathway.

Adiponectin (APN), a circulating adipose-derived hormone that regulates inflammation and energy metabolism, has beneficial effects on the cardiovascular disorders. Serum APN levels are lower in patients with coronary artery disease and higher in patients with chronic kidney disease. However, the precise role of APN in acute reno-vascular disease is not clear. Results of the present study show that serum APN concentration decreased after renal ischemia reperfusion (I/R) injury in mice. In addition, I/R-induced renal dysfunction (elevated serum creatinine and urea levels), inflammation (number of infiltrating neutrophils, myeloperoxidase activity), and apoptotic responses (apoptotic cell number and caspase-3 activation) were attenuated in APN-treated compared to control mice. Molecular and biochemical analysis revealed that APN up-regulates heme oxygenase-1 (HO-1) via peroxisome-proliferator-activated-receptor-α (PPARα) dependent pathway which is mediated through the enhancement of COX-2 and 6-keto PGF1α expression. Chromatin immune-precipitation assay demonstrated that APN increases the binding activity of PPARα to PPRE region of HO-1 promoter. Furthermore, APN induced HO-1 expression was only found in wild-type but not in PPARα gene deleted mice. This provides in vivo evidence that APN mediated HO-1 expression depends on PPARα regulation. In conclusion, our results provide a novel APN mediated prostacyclin-PPARα-HO-1 signaling pathway in protecting renal I/R injury.

In vivo therapy of myocardial infarction with mesenchymal stem cells modified with prostaglandin I synthase gene improves cardiac performance in mice.

AIM: Intra-myocardial injection of adult bone marrow-derived stem cells (MSC) has recently been proposed as a therapy to repair damaged cardiomyocytes after acute myocardial infarction (AMI). PGI(2) has vasodilatation effects; however, the effects of combining both MSC and PGI(2) therapy on AMI have never been evaluated. MAIN METHODS: We genetically enhanced prostaglandin I synthase (PGIS) gene expression in mouse mesenchymal stem cells (MSC) using lentiviral vector transduction (MSC(PGIS)). Mice were subjected to an AMI model and injected (intra-myocardially) with either 5×10(4) MSCs or MSC(PGIS) before surgery. Fourteen days post AMI, mice were analyzed with echocardiography, immunohistochemistry, and apoptotic, and traditional tissue assays. KEY FINDINGS: Lenti-PGIS transduction did not change any characteristic of the MSCs. PGIS over-expressed MSCs secreted 6-keto-PGF1α in the culture medium and decreased free radical damage during hypoxia/re-oxygenation and H(2)O(2) treatment. Furthermore, splenocyte proliferation was significantly suppressed with MSC(PGIS) as compared with MSCs alone. Fourteen days post AMI, echocardiography showed more improvement in cardiac function of the MSC(PGIS) group than the MSC alone group, sham-operated group, or artery ligation only group. The histology of MSC(PGIS) treated hearts revealed MSCs in the infarcted region and decreased myocardial fibrosis/apoptosis with limited cardiac remodeling. Furthermore, the level of the vascular endothelial growth factor was elevated in the MSC(PGIS) group as compared to the other three groups. SIGNIFICANCE: In summary, our results provide both in vitro and in vivo evidence for the beneficial role of MSC(PGIS) in limiting the process of detrimental cardiac remodeling in a mouse AMI model during early stages of the disease.

The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes.

Peroxisome-proliferator-activated receptor γ (PPARγ) plays a critical role in regulation of adipocyte differentiation and insulin sensitivity. To become functional, PPARγ must be activated by binding an appropriate ligand. Polyunsaturated fatty acids (PUFA) are potential ligands for PPARγ. The current experiment was designed to determine the potential for PUFA, particularly eicosapentaenoic acid and docosahexaenoic acid, to activate the function of porcine PPARγ in vivo. Transgenic mice, expressing porcine PPARγ in skeletal muscle were generated and fed with a high-saturated fat (beef tallow) or high-unsaturated fat (fish oil) diet for 4 months. When transgenic mice were fed a fish oil supplemented diet, the expression of adipogenic and glucose uptake genes was increased, leading to reduced plasma glucose concentration. The PPARγ transgene increased the expression of Glut4 in the muscle. This result suggests that there was increased glucose utilization and, therefore, a reduced blood glucose concentration in the transgenic mice. Also, the plasma adiponectin was elevated by fish oil treatment, suggesting a role of adiponectin in mediating the PUFA effect. These results suggest that PUFA may serve as a natural regulator of glucose uptake in vivo and these effects are mainly through PPARγ function.

Thermosensitive chitosan-gelatin-glycerol phosphate hydrogels as a cell carrier for nucleus pulposus regeneration: an in vitro study.

Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the gelatin molecules were added to the thermosensitive chitosan/beta-glycerol phosphate (C/GP) disodium salt hydrogels to form chitosan/gelatin/beta-glycerol phosphate (C/G/GP) disodium salt hydrogels which were applied as a cell carrier for nucleus pulposus (NP) regeneration. The gelation temperature, gelation time, and gel strength of the C/G/GP hydrogels were analyzed by the rheometer. NP cells were then harvested from the intervertebral discs of the adult New Zealand white rabbits and cultured in monolayer or in C/G/GP hydrogel, respectively. The cell viability, material-mediated cytotoxicity, cell proliferation, production of sulfated glycosaminoglycans, anabolic/catabolic gene expressions, and extracellular matrix-related gene expressions of the NP cells were demonstrated. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1-33.8 degrees C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. Among those, C/GP with 1% gelatin addition showed the most promising gelation time and gel strength and were utilized in the later experiments. From the results of cell activity, cytotoxicity, and cell proliferation assays, NP cells cultured in C/G/GP hydrogel had normal cell viability and cell proliferation that indicated the hydrogel was noncytotoxicity. The amounts of sulfated glycosaminoglycans of NP cells cultured in C/G/GP hydrogels were significantly higher than monolayer cultured. Considering the extracellular matrix-related gene expression, type II collagen and aggrecan of NP cells cultured in the hydrogels greatly increased than those in monolayer culture. On the contrary, the unfavorable gene expression, such as that of type I collagen, was decreased significantly. The results reveal that gelatin added into C/GP hydrogel significantly shortened the gelation time and improved the gel strength without influencing the biocompatibility. NP cells cultured in the C/G/GP hydrogel also displayed better gene expressions when compared with the monolayer culture. This study indicates that using chitosan/gelatin hydrogel for NP cell culture is feasible and may apply in minimal invasive intervertebral disc surgery in the future.

Insulin regulates the expression of adiponectin and adiponectin receptors in porcine adipocytes.

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by two subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of insulin on the expression of adiponectin and its receptors. We demonstrated that in the presence of 10 nM insulin, addition of 1 microM of insulin or rosiglitazone (a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist) had no effect on the expression of adiponectin and AdipoR genes in differentiated porcine adipocytes. However, the addition of 1 microM insulin plus 1 microM rosiglitazone significantly increased the AdipoR2 mRNA in differentiated porcine adipocytes. Using the phosphatidylinositol 3-kinase inhibitor (PI3K inhibitor, LY 294002), we found that insulin inhibited the expression of AdipoR2 through the PI3K pathway and this inhibition was blocked by addition of rosiglitazone. When porcine adipocytes were cultured without insulin, supplementation with 10 nM insulin inhibited the expression of AdipoR2 and this inhibition effect was also blocked by addition of rosiglitazone. Therefore, these data suggest that a PPARgamma agonist increases expression of AdipoR2 and that insulin inhibits the expression of AdipoR2 through the PI3K pathway.

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis.

BACKGROUND: Prolactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma. RESULTS: The expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine. CONCLUSION: Our results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Differentially expressed transcripts in shell glands from low and high egg production strains of chickens using cDNA microarrays.

We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

Proliferin enhances microvilli formation and cell growth of neuroblastoma cells.

Proliferins (also termed mitogen-regulated proteins; MRP/PLFs) belong to the prolactin gene family. Mrp/Plfs are involved in angiogenesis of the uterus and placenta and maximally expressed during midgestation and decline through the remainder of the gestation period in mouse placenta. The tissue expressions of Mrp/Plfs are mainly documented in placenta, hair follicles of skin and in wound healing. In this report, we demonstrate that Plf1, Plf1 minus exon3, Plf2 and Mrp3 but not Mrp4 are expressed in mouse whole brain by diagnostic RT-PCR and Western blotting. The expression levels of Mrp/Plf mRNAs in mouse brains were low during the neonatal period, but higher in embryonic and adult stages, indicating Mrp/Plfs expression profiles are different in mouse brain and placenta. Interestingly, endogenous Mrp/Plfs were detected using immunostaining both in mouse brain sections and the neuroblastoma cell line, Neuro-2a cells. The function of PLF1 was explored by expressing exogenous PLF1 in Neuro-2a cells. This resulted in increased microvilli. Neuro-2a cells with stable expression of PLF1 had increased proliferation compared with normal and stable expressing EGFP cells when cell reached saturation density. Together these data, strongly suggest that MRP/PLFs mediate microvilli formation and contribute to cell proliferation of neuroblastoma cells.