Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal Rat Calvaria Cells in an Oxidative Stress Environment.

Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for 4 days (D5-9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (P < 0.05). This suppression was partially blocked when cells were pretreated 12 h with 20 µM QRC. Hydrogen peroxide also produced sustained up-regulation of heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC), which was partially blocked in hydrogen peroxide-treated cells that first received QRC pretreatment. The alterations in the antioxidant stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. J. Cell. Physiol. 231: 2779-2788, 2016. © 2016 Wiley Periodicals, Inc.

Quercetin Metabolites Up-Regulate the Antioxidant Response in Osteoblasts Isolated From Fetal Rat Calvaria.

Oxidative stress contributes to osteoporosis by suppressing differentiation of osteoblasts, suggesting the osteoblast antioxidant response may be a viable strategy for osteoporosis prevention. Quercetin, an antioxidant flavonol, up-regulates the antioxidant response in many cell types, but studies are needed to understand the effects of quercetin plasma metabolites on the osteoblast antioxidant response. The first specific aim was to examine antioxidant response genes and proteins in osteoblasts exposed to plasma quercetin metabolites. The second specific aim was to identify potential signaling pathways in the osteoblast antioxidant response that mediate the effect of quercetin, specifically Nrf2, ERK1/2, and NFκB p65. Osteoblasts isolated from fetal rat calvaria were treated with doses up to 20 µM of three different quercetin metabolites found in blood plasma after consumption of quercetin-rich foods or supplements: quercetin aglycone (QRC), isorhamnetin (ISO), or quercetin 3-O-glucuronide (Q3G). Alternatively, some cells received a 2:1:1 mixture of all three metabolites (10 µM Q3G: 5 µM ISO: 5 µM QRC) to evaluate synergistic effects. Antioxidant response genes and proteins known to be up-regulated by quercetin were analyzed along with Nrf2, ERK1/2, and NFκB proteins. Both QRC and ISO, but not Q3G, up-regulated heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC) at the mRNA and protein level. Synergistic effects of metabolites were not observed. Up-regulation of HO-1 and GCLC was associated with suppression of phosphorylated ERK1/2 and NFκB, but no alterations in Nrf2 protein levels were observed. This study shows that the antioxidant response of osteoblasts is differentially stimulated by quercetin metabolites.

Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes.

BACKGROUND: With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., ß-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. CONCLUSION/SIGNIFICANCE: Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.

Helenalin-mediated post-transcriptional regulation of p21(Cip1) inhibits 3T3-L1 preadipocyte proliferation.

We have previously shown that post-transcriptional mechanisms involving the 26S proteasome regulate the cyclin-dependent kinase inhibitors (CKIs), p21(Cip1) and p27(Kip1) during preadipocyte proliferation. Earlier studies further demonstrated that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 accumulation, an F-box protein mediating SCF E3 ligase ubiquitylation and degradation of both CKIs during S phase progression. Data presented here demonstrate that helenalin dose-dependently induced G1 arrest of synchronously replicating 3T3-L1 preadipocytes. This effect occurred in the absence of discernable indices of cell toxicity or apoptosis under the conditions used in this study. Our results demonstrate that helenalin markedly increased p21 protein accumulation in both density-arrested and proliferating preadipocytes in a dose-dependent manner. This increase in p21 protein abundance occurred without change in mRNA transcript demonstrating that post-transcriptional mechanisms were involved. This notion was further supported by the modest accumulation of polyubiquitylated p21 following treatment with helenalin suggesting that suppression of targeted p21 proteolysis by the 26S proteasome contributed to helenalin-mediated p21 accumulation. The increase in p21 protein was compartmentalized to the nucleus where p21 is known to inhibit cell cycle progression. Finally, helenalin increased protein-protein interactions between p21 and cyclin-dependent kinase 2 (Cdk2) which may account in part for the anti-proliferative effect in 3T3-L1 preadipocytes.

Novel effect of helenalin on Akt signaling and Skp2 expression in 3T3-L1 preadipocytes.

We have previously shown that the F-box protein, Skp2, is highly regulated during preadipocyte proliferation and plays a mechanistic role in p27 degradation during cell cycle progression. Data presented here demonstrate that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 protein accumulation during early phases of 3T3-L1 adipocyte differentiation. Furthermore, helenalin was shown to completely block p27 degradation, cyclin A accumulation, and G(1)/S transition resulting in G(1) arrest. Helenalin was also shown to block Skp2 mRNA accumulation in a concentration-dependent manner and to completely suppress hormonally induced Skp2 promoter activity suggesting transcriptional mechanisms were involved. Examination of signaling events previously determined to be important for Skp2 upregulation during adipogenesis revealed impaired Akt phosphorylation immediately preceding the inhibitory effect of helenalin on Skp2 mRNA accumulation. These studies demonstrate a novel effect of helenalin on Skp2 regulation and growth factor receptor signaling during early stages of adipocyte differentiation.

Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate.

The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA.

Increased GADD153 gene expression during iron chelation-induced apoptosis in Jurkat T-lymphocytes.

Depriving cells of iron likely stresses them and can result in cell death. To examine the potential relationship between this form of stress and cell death, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). Such treatment produced evidence of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting involvement of caspases in initiating apoptosis. Indeed, a selective caspase-3 inhibitor prevented the effects of DFO. During the early induction period of apoptosis, GRP78 and HSP70 mRNA expression was not affected. In contrast, there was mainly increased mRNA expression of Growth Arrest and DNA Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Furthermore, fortifying cells with antioxidants did not prevent the increased GADD153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither reactive oxygen species nor DNA damage was involved in triggering GADD153 gene activation. DFO also caused GADD153 protein to be expressed. Because GADD153 is recognized as a pro-apoptotic gene, these findings generate the notion that GADD153 might help mediate apoptosis in iron-deficient cells.

Induction of GADD gene expression by phenethylisothiocyanate in human colon adenocarcinoma cells.

Phenethylisothiocyanate (PEITC), a potential cancer chemopreventive agent, induces colon cancer cell death, but the mechanism is not entirely clear. Therefore, the aim of this study was to further clarify the molecular effects of PEITC in causing death of human colon adenocarcinoma cells. When incubated with PEITC, HCT-116 colonocytes showed morphological features characteristic of apoptosis, such as irregular cell shape, translocation of plasma membrane phosphatidylserine, and also chromatin condensation and fragmentation. These changes occurred after single-strand breaks in DNA were detected, suggesting that PEITC induced irreparable DNA damage, which in turn triggered the process of apoptosis. DNA macroarray analysis of a selected small cluster of apoptosis-related genes revealed noticeably higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. This led to investigation of other GADD gene members possibly affected by PEITC. Whereas GADD34 mRNA expression increased just slightly, there was an appreciable elevation of the mRNA for GADD153, which is recognized as a pro-apoptotic gene. The effect of PEITC on GADD153 was attenuated by either actinomycin D or N-acetylcysteine, suggesting that PEITC-induced upregulation of GADD153 mRNA expression was partly at the level of transcriptional activation involving reactive oxygen species. Additionally, PEITC-induced upregulation of GADD153 mRNA expression did not appear to require p53, based on the observation that PEITC also increased GADD153 mRNA expression in HCT-15 colonocytes, which are known to express mutant p53. These findings suggest that PEITC creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis.

Safety assessment of recombinant green fluorescent protein orally administered to weaned rats.

Several proposed biotechnological applications of green fluorescent protein (GFP) are likely to result in its introduction into the food supply of domestic animals and humans. We fed pure GFP and diets containing transgenic canola expressing GFP to young male rats for 26 d to evaluate the potential toxicity and allergenicity of GFP. Animals (n = 8 per group) were fed either AIN-93G (control), control diet plus 1.0 mg of purified GFP daily, modified control diet with 200 g/kg canola (Brassica rapa cv Westar), or control diet with 200 g/kg transgenic canola containing one of two levels of GFP. Ingestion of GFP did not affect growth, food intake, relative weight of intestine or other organs, or activities of hepatic enzymes in serum. Comparison of the amino acid sequence of GFP to known food allergens revealed that the greatest number of consecutive amino acid matches between GFP and any food allergen was four, suggesting the absence of common allergen epitopes. Moreover, GFP was rapidly degraded during simulated gastric digestion. These data indicate that GFP is a low allergenicity risk and provide preliminary indications that GFP is not likely to represent a health risk.