Strand cDNA Synthesis Research Articles

Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori, Dually Couples to the Gq and Gs Signaling Cascades

Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca(2+) mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Capabilities and Challenges of Examination of Gene Expression for Quality Assessment of Domestic Cat Embryos

Early embryos are characterized by an accurately controlled gene expression pattern that might be deregulated during in vitro culture (IVC). The expression pattern of the developmental genes may serve as markers for embryo quality. Here, we examined the temporal pattern of relative mRNA abundance of genes important for early embryonic development in embryos produced by different fertilization methods [in vitro fertilization (IVF) vs intracytoplasmic sperm cell injection (ICSI)] and sperm sources (fresh vs frozen-thawed) applying reverse transcriptase (RT) PCR. The temporal pattern of gene expression was found to be gene specific and similar in all four examined groups in a semi-quantitative assay. In morulae, higher relative mRNA levels were found in embryos generated with fresh sperm, whereas in blastocysts, mRNA abundance tended to be higher in embryos produced with cryopreserved sperm cells. This indicates an influence of sperm cryopreservation on the temporal gene expression pattern in early cat embryos. We also examined relative mRNA abundances by real-time quantitative RT-PCR in blastocysts. In this context, blastocysts produced with fresh semen tended to have lower DNA methyltransferase 3A (DNMT3A) but higher gap junction protein alpha 1 (GJA1) and octamer-binding transcription factor 4 (OCT4) mRNA levels compared with those derived with frozen-thawed semen. We conclude that assessing embryo quality by measuring gene expression pattern in early embryos is challenging because of a high variability between individual embryos.

Expression profile of ghrelin and ghrelin receptor in cyclic goat ovary

Ghrelin, a 28-amino acid acylated peptide, was identified as the endogenous ligand for the GH secretagogue receptor and recently the role of ghrelin in reproduction was established. Total RNA was extracted from corpus luteum (CL) of the slaughterhouse derived goat ovaries of different stages of cyclicity. First strand cDNA synthesis was done using constant quantity of total RNA (8µg / 20 µl reaction mixture) and M-MuLV-RT at 36°C for 1h. Semi quantitative PCR analysis as well as real time PCR (Q-PCR) analysis revealed, persistent expression of ghrelin and its receptor throughout the estrous cycle. Ghrelin mRNA corresponded the activity of CL i.e. minimum expression in the degenerating CL and it was at peak in the fully grown CL and similar trend was also observed for ghrelin receptor. Further, ghrelin and its receptor was absent in prepubertal ovary while it was present in cyclic goat ovaries. It can be concluded that ghrelin may have role in the regulation of ovarian follicular development and early embryonic development.

Protein Kinase Cα (PKCα) Regulates p53 Localization and Melanoma Cell Survival Downstream of Integrin αv in Three-dimensional Collagen and in Vivo

Protein kinase C α (PKCα) is overexpressed in numerous types of cancer. Importantly, PKCα has been linked to metastasis of malignant melanoma in patients. However, it has been unclear how PKCα may be regulated and how it exerts its role in melanoma. Here, we identified a role for PKCα in melanoma cell survival in a three-dimensional collagen model mimicking the in vivo pathophysiology of the dermis. A pathway was identified that involved integrin αv-mediated up-regulation of PKCα and PKCα-dependent regulation of p53 localization, which was connected to melanoma cell survival. Melanoma survival and growth in three-dimensional microenvironments requires the expression of integrin αv, which acts to suppress p53 activity. Interestingly, microarray analysis revealed that PKCα was up-regulated by integrin αv in a three-dimensional microenvironment-dependent manner. Integrin αv was observed to promote a relocalization of endogenous p53 from the nucleus to the cytoplasm upon growth in three-dimensional collagen as well as in vivo, whereas stable knockdown of PKCα inhibited the integrin αv-mediated relocalization of p53. Importantly, knockdown of PKCα also promoted apoptosis in three-dimensional collagen and in vivo, resulting in reduced tumor growth. This indicates that PKCα constitutes a crucial component of the integrin αv-mediated pathway(s) that promote p53 relocalization and melanoma survival.

Tumor IGF-1 expression as a predictive biomarker for IGF1R-directed therapy in advanced pancreatic cancer (APC).

4054 Background: IGF-1 up-regulates PC proliferation and invasiveness through activation of PI3K/Akt signaling pathway and down-regulates PTEN. We investigated IGF-1 expression in tissue and blood as potential predictive markers in phase II study of IGF1R-directed monoclonal antibody, MK-0646 in APC. Prior phase I established the MTD of MK0646 at 5 mg/kg with gemcitabine (G) and erlotinib (E) and 10 mg/kg with G alone. Methods: Patients (pts) with stage IV, previously untreated APC, ECOG PS 0-1, adequate hematologic and organ function were enrolled. Arm A: G 1,000 mg/m2 over 100 min, weekly x 3, MK-0646 weekly x 4; Arm B: G 1000 mg/m2 and MK-0646 + E 100 mg daily. Arm C (control) was G 1,000 mg/m2 + E 100 mg. Cycles were repeated every 4 weeks. Pts were equally randomized in the 3 arms. Primary study objective was progression-free survival (PFS). Pre-treatment peripheral blood samples were measured for IGF-1 level by ELISA; archival core biopsies were analyzed for IGF-1 mRNA expression. RNA extraction from FFPE samples used Roche Transcriptor First Strand cDNA Synthesis Kit. TaqMan PreAmp technique was used to amplify target cDNA prior to TaqMan RT-PCR analysis. Cox proportional hazards model for PFS analyzed the interaction between tissue IGF-1 expression and treatment. Results: 50 pts were enrolled (A=15, B=16,C=16 pts, 3 ineligible). Median PFS of arms A, B and C were 5.5 months (95% CI: 3.9 – NA), 3.0 months (95% CI:1.8 – 5.6) and 2.0 months (95% CI: 1.8 – NA), respectively (log-rank test; p = 0.17). Median OS of A was 11.3 months (95% CI: 8.9 – NA), B 8.9 months (95% CI: 5.3 – NA) and C 5.7 months (95% CI: 2.0 – NA) (log-rank test; p = 0.44). 35 archival core biopsies were analyzed, 21 had adequate tissue for analysis. Using a Multivariable Cox proportional hazards model for PFS, where IGF-1 was dichotomized at the median, there was a 76% reduction in the risk of disease progression or death in arm A as compared with the control (arm C) at high IGF-1 level (p = 0.16). When IGF-1 was fitted as a continuous variable, this reduction was 96% (p = 0.08). There was no correlation between tissue and serum IGF-1. Conclusions: Tissue expression of IGF-1 level may represent a promising predictive biomarker for IGF1R-directed therapy in APC.

Abstract 1155: A discriminatory set of transcripts, suitable for differentiation of normal, colorectal cancer and adenoma cases from both fresh frozen biopsy and formalin-fixed, paraffin-embedded (FFPE) tissue samples - potential biomarkers of colorectal dyplasia-carcinoma transition

Abstract Background and aims: Though histopathology is still the gold standard for differentiating stages of colorectal disease progression, the cut slide may represent only a fragment of the whole sample and disease features, such as dysplastic areas of flat adenomas, may remain hidden due to inappropriate sampling or tissue orientation. The early molecular detection of the colorectal dysplasia-carcinoma transition may enhance the strenght of diagnosis from colonic biopsies. Using high-throughput microarray-based biomarker screening low cost diagnostic array real-time PCR panels may be developed. We aimed to determine molecular markers for enhancing the differentiation of high-grade dysplasia from colorectal carcinoma (CRC) and to analyze the applicability of FFPE tissue samples. Material and Methods: Discriminatory transcript set was identified using HGU133plus2 microarrays (Affymetrix Inc.) on 53 biopsy samples (22 CRC, 20 adenoma, 11 normal). For testing the classificatory power of the discriminatory genes, 94 independent biopsies (27 CRC, 29 adenoma, 38 normal) were analyzed on microarrays. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma, 20 normal) using LightCycler®480 system (Roche) and RealTime ready assays. Reverse transcription was done using Transcriptor First Strand cDNA Synthesis Kit (Roche). For testing the applicability of FFPE tissues, total RNA was isolated from 10um-thick slides of 5 CRC and 2 adjacent normal samples using High Pure RNA Paraffin Kit (Roche). The same cDNA synthesis and real-time PCR conditions were applied as in case of fresh frozen samples. Results: A set of 11 transcripts was determined which could correctly discriminate between not only the normal, adenoma and CRC samples, but between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent fresh frozen biopsy samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples were correctly classified in discriminant analysis. According to the RT-PCR results for the set of 11 markers, tumorous and normal FFPE tissue samples could be distinguished by 100% sensitivity and specificity. Conclusion: The identified transcripts could correctly characterize the dysplasia-carcinoma transition in colonic tissue samples, also on a large independent sample set and on FFPE tissue samples. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic panels can become part of the automated routine procedure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1155. doi:1538-7445.AM2012-1155

Abstract 1706: Comparison of automated and manual RNA isolation from stabilized peripheral blood samples (PBL) for expression analysis of colorectal cancer patients

Abstract Background and aims: PBL based molecular diagnostics requires automated sample processing and evaluation of the isolated RNA for RT-PCR analysis. Our aim was to compare the automated PBL RNA isolation on MagNA Pure 96 with conventional manual RNA isolation by the PAXgene Blood RNA kit and to determine the purity, recovery, RNA integrity (RIN) and sensitivity of downstream RT-PCR applications. Materials and Methods: PBL samples were taken from 25 CRC and 19 healthy normal patients into PAXgene Blood RNA Tubes (PreAnalytix). Total RNA was manually isolated using the PAXgene Blood RNA Kit (Qiagen) for 2 blood samples of each patient. In parallel, the MagNA Pure 96 Cellular RNA Kit LV and MagNA Pure 96 instrument (MP96) (Roche) were applied for total RNA extraction from the other 2 blood samples of each patient. Total RNA concentration was measured using a NanoDrop spectrophotometer. RIN was determined on a Bioanalyzer 2100 (Agilent) using the RNA6000 Nano Kit. Reverse transcription was carried out from 1ug total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). The expression of 6 CRC markers from previous microarray analysis (ASGR2, CLIP1, ARHGAP18, 1569263_at, C19orf43, MALAT1) was determined by quantitative real-time PCR on LightCycler 480 System (Roche). Results: The MP96 isolation resulted in higher RNA yields (MP96: 5.99±2.04 ug/tube; manual: 5.65±2.75 ug/tube). The purity OD260/280 of manually isolated samples (2.09 ± 0.03) was equal to that of MP96 isolated ones (2.01 ± 0.05), all samples fulfilled the requirement. The OD230/260 was significantly better in case of MP96 isolated samples (1.85 ± 0.23) compared to manually isolated ones (1.52 ± 0.57) (p<0.0001). The RNA integrity was acceptable in both isolation method, however the manual protocol provided more intact RNA with slightly higher RNA integrity numbers (RIN manual: 9,19 ± 0,26; RIN MP96: 8,25 ± 0,35; p<0.0001). No significant RIN differences were detected between prospective and archived samples. Comparison of both extraction methods and evaluation by RT-PCR showed a linear regression (r = 0.98). In real-time PCR experiments, ARHGAP18 (spec.:80%, sens.:75%) and ASGR2 (spec.:70%, sens.:75%) were proved to be the best differentiating individual markers. The CTs of the RNA extracted by MP96 were lower than those of the manual extraction (means higher expression) reflecting the higher mRNA yield/ratio of the MP96 method. Conclusion: Automated RNA isolation from stabilized PBL samples on MP96 device provides a high quality, purity and yield of total RNA. The CTs of the RNA extracted by MP96 were better than those of the manual extraction. The MP96 extraction is a fully automated, fast and reliable method with 96 RNA extractions within 85 min, compared to the equally precise, but time consuming manual extraction by the PAXgene Blood RNA kit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1706. doi:1538-7445.AM2012-1706

The Role of Brain Derived Neurotrophic Factor in Depression In a Mouse Model

BackgroundBrain derived neurotrophic factor(BDNF) levels have been thought to be a link to depression. It is not clear in which brain region BDNF mRNA levels are reduced in BDNF‐KIV mice. While BDNF gene expression is regulated by at least 9 promoters, promoter IV is of interest because it is suggested to be decreased in depressed patients.ObjectiveIn our research, we generated mutant mice that lack promoter IV‐driven BDNF expression and show depression‐like behavior (BDNF‐KIV). We aimed to determine 1) which brain regions show substantially decreased total BDNF mRNA levels, and 2) whether there is a correlation between brain and blood BDNF levels using the mouse model of depression. We also screened for what gene expression was affected by lack of promoter IV‐driven BDNF expression.MethodsMouse RNA was extracted from various neuroanatomical structures of the brain as well as PBMC cells in the blood using QIA‐Shredder and RNeasy column systems. A cDNA was prepared using the Transcriptor First Strand cDNA Synthesis Kit and a sample was preamplified using PreAmp Master Mix. A primer‐probe plate was designed to express genes, focusing on the BDNF exons. Gene expression was calculated with a reference gene, Cyclophilin D. The Biomark HD system by Fluidigm expressed the genes using qRT‐PCR, comparing BDNF levels with other genes in WT versus BDNF‐KIV mice.ResultsGene expression showed BDNF levels were lower in brain tissues of KIV mice than in WT mice, especially in the prefrontal cortex and hippocampus. BDNF levels were higher in PBMC cells of KIV mice in comparison with WT mice. The results were calculated directly from the gene expression in Exon IX.ConclusionBDNF levels are linked to depression and are found to be lower in KIV mice, especially in the PFC and the HIP. However, BDNF levels are reversed in PBMC cells, suggesting that BDNF levels in PBMC do not correlate to those in the brain and cannot be used as a biomarker of depression.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Axonal Neuropathy-associated TRPV4 Regulates Neurotrophic Factor-derived Axonal Growth

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Low-Dose-Rate, Low-Dose Irradiation Delays Neurodegeneration in a Model of Retinitis Pigmentosa

The existence of radiation hormesis is controversial. Several stimulatory effects of low-dose (LD) radiation have been reported to date; however, the effects on neural tissue or neurodegeneration remain unknown. Here, we show that LD radiation has a neuroprotective effect in mouse models of retinitis pigmentosa, a hereditary, progressive neurodegenerative disease that leads to blindness. Various LD radiation doses were administered to the eyes in a retinal degeneration mouse model, and their pathological and physiological effects were analyzed. LD gamma radiation in a low-dose-rate (LDR) condition rescues photoreceptor cell apoptosis both morphologically and functionally. The greatest effect was observed in a condition using 650 mGy irradiation and a 26 mGy/minute dose rate. Multiple rounds of irradiation strengthened this neuroprotective effect. A characteristic up-regulation (563%) of antioxidative gene peroxiredoxin-2 (Prdx2) in the LDR-LD-irradiated retina was observed compared to the sham-treated control retina. Silencing the Prdx2 using small-interfering RNA administration reduced the LDR-LD rescue effect on the photoreceptors. Our results demonstrate for the first time that LDR-LD irradiation has a biological effect in neural cells of living animals. The results support that radiation exhibits hormesis, and this effect may be applied as a novel therapeutic concept for retinitis pigmentosa and for other progressive neurodegenerative diseases regardless of the mechanism of degeneration involved.

Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria

Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

The Histone Modifications Governing TFF1 Transcription Mediated by Estrogen Receptor

Transcription regulation by histone modifications is a major contributing factor to the structural and functional diversity in biology. These modifications are encrypted as histone codes or histone languages and function to establish and maintain heritable epigenetic codes that define the identity and the fate of the cell. Despite recent advances revealing numerous histone modifications associated with transcription regulation, how such modifications dictate the process of transcription is not fully understood. Here we describe spatial and temporal analyses of the histone modifications that are introduced during estrogen receptor α (ERα)-activated transcription. We demonstrated that aborting RNA polymerase II caused a disruption of the histone modifications that are associated with transcription elongation but had a minimal effect on modifications deposited during transcription initiation. We also found that the histone H3S10 phosphorylation mark is catalyzed by mitogen- and stress-activated protein kinase 1 (MSK1) and is recognized by a 14-3-3ζ/14-3-3ε heterodimer through its interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH. We showed that H3S10 phosphorylation is a prerequisite for H3K4 trimethylation. In addition, we demonstrated that SET8/PR-Set7/KMT5A is required for ERα-regulated transcription and its catalyzed H4K20 monomethylation is implicated in both transcription initiation and elongation. Our experiments provide a relatively comprehensive analysis of histone modifications associated with ERα-regulated transcription and define the biological meaning of several key components of the histone code that governs ERα-regulated transcription.

Letter to the editor] Application of linear polyacrylamide coprecipitation of denatured templates for PCR amplification of ultra-rapidly reannealing DNA

Letter to the editor] Application of linear polyacrylamide coprecipitation of denatured templates for PCR amplification of ultra-rapidly reannealing DNA

Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.