McCoy's 5A Medium Research Articles

RhoGDI SUMOylation at Lys-138 Increases Its Binding Activity to Rho GTPase and Its Inhibiting Cancer Cell Motility

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Effects of Basal Media and Supplements on Diethylstilbestrol‐Treated Immature Mouse Primary Granulosa Cell Growth and Regulation of Steroidogenesis In Vitro

The objectives of this study were to investigate the effects of basal media, supplements, including mixture of 5 mg/ml insulin, 5 mg/ml transferrin and 5 g/ml selenium (ITS), bovine serum albumin (BSA), sodium pyruvate (PNa) and epidermal growth factor (EGF) compared with serum on the proliferation of diethylstilbestrol-treated C57BL/6J mouse primary granulosa cells (GCs) to develop a serum-free culture system and to use this system to evaluate the effects of these supplements on GCs' processes of steroidogenesis and related regulation mechanism. DMEM/F12, McCoy's 5a, RPMI Medium 1640, Medium 199 and a-MEM were chosen as basal media, and McCoy's 5a proliferated the GCs greatly (p < 0.05) with a better attachment and morphology. Granulosa cells showed a better proliferation status in serum-containing media than serum-free media, BSA, ITS, PNa and EGF increased the proliferation of GCs in serum-free system gradually, while the latter three had the same effect on supporting GCs' proliferation with serum. After 2-day culture, the cells cultured remained P450arom positive, which sustained a normal morphology and there was no sign of apoptosis when supplements were added in the serum-free condition. Only ITS induced oestradiol production, while ITS, BSA and PNa enhanced progesterone secretion significantly indicating the promotion of differentiation. And these supplements functioned partly through increasing the transcription level of StAR, 3β-HSD, P450scc or P450arom. In conclusion, McCoy's 5a medium was a better basal medium, and ITS and PNa promoted both the proliferation and differentiation of primary GCs in serum-free culture system, showing a satisfactory state for GCs' physiological study.

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics.

Peroxiredoxin II Restrains DNA Damage-induced Death in Cancer Cells by Positively Regulating JNK-dependent DNA Repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.

TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

Effects of Daidzein on mRNA Expression of Bone Morphogenetic Protein Receptor Type I and II Genes in the Ovine Granulosa Cells

Daidzein, a natural isoflavonoid phytoestrogen, structurally resembles estradiol (E2) and possesses estrogenic activity. This study was designed to test the hypothesis that daidzein may mimic the effects of E2 on ovine follicle development by regulation of the mRNA expression of bone morphogenetic protein receptor genes and thereby influence the reproductive system. Granulosa cells were cultured in serum-free McCoy's 5A medium with and without supplementation of daidzein. Results showed that daidzein (10-100 ng/ml) significantly increased the proliferation of ovine granulosa cells (p

Protective Effect of Quercetin on the Reproductive Toxicity of 4-nitrophenol in Diesel Exhaust Particles on Male Embryonic Chickens

The 4-nitrophenol (PNP) in diesel exhaust particles (DEP) has been identified as a vasodilator and is a known degradation product of the insecticide parathion. In this study, the protective effect of quercetin, a potent oxygen free radical scavenger and metal chelator, against the oxidative damage of PNP on cultured testicular cells was studied in male embryonic chickens. Testicular cells from Day 18 embryos were cultured in serum-free McCoy's 5A medium and challenged with quercetin (1.0 microg/ml) alone or in combinations with PNP (10(-7)-10(-5) M) for 48 h. The oxidative damage was estimated by measuring cell viability, content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidation (GSH-Px) activity. The results showed that exposure to PNP (10(-5) M) induced condensed nuclei, vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNP induced lipid peroxidation by elevation of the content of MDA. Exposure to PNP also decreased GSH-Px activity and SOD activity. However, simultaneous supplementation with quercetin restored these parameters to the same levels as the control. Consequently, PNP induced oxidative stress in spermatogonial cells, and dietary quercetin may attenuate the reproductive toxicity of PNP to restore the intracellular antioxidant system in the testicular cells of embryonic chickens.

Assembly of osteoblastic cell micro-arrays on diamond guided by protein pre-adsorption

We investigate the role of fetal bovine serum (FBS) proteins in the assembly of osteoblastic cells (SAOS-2) in McCoy's 5A medium on synthetic monocrystalline and nanocrystalline diamonds having H- and O-terminated array patterns. We characterize i) adhesion of proteins and cells to diamond by shaking experiments (1000 rpm), fluorescence, and atomic force microscopy, ii) distribution of FBS proteins by micro-Raman spectroscopy, and iii) influence of pre-adsorbed specific FBS proteins (albumin, fibronectin, vitronectin) on the cell assembly. There is 40% lower adhesion of cells on H-terminated diamond with adsorbed FBS compared to this surface without FBS as well as to O-terminated surfaces. Spatially resolved Raman spectroscopy of C―H stretching bands around 2900 cm − 1 showed uniform coverage of H/O-diamond by the albumin in more than 50 nm thick FBS layers. The other FBS proteins were not detectable by Raman. Pre-adsorption of the specific proteins prior to cell plating indicated that the cell preference to O-terminated diamond is controlled by the fibronectin rather than by the albumin. We discuss the data with a view to the cell preferential assembly on H/O-diamond micro-arrays.

CVD diamond films with hydrophilic micro-patterns for self-organisation of human osteoblasts

Nanocrystalline diamond (NCD) films were prepared by microwave plasma-enhanced chemical vapour deposition (CVD) on Si substrates of different roughness (1 and 500 nm). Diamond nano-crystals are up to 50 nm in size and RMS surface roughness is less than 20 nm. The NCD films were cleaned chemically and terminated by hydrogen using plasma treatment (800 °C, 10 min) to generate a hydrophobic surface. Photolithography mask and oxygen plasma (300 W r.f. power, 3 min) were used to generate O-terminated (hydrophilic) patterns (30–200 μm wide) separated by a H-terminated (hydrophobic) surface. Osteoblast-like human cells were seeded on the patterned flat and rough NCD films in McCoy's 5A medium supplemented with 15% fetal bovine serum (FBS). After two days incubation the cells preferentially adhered on the O-terminated stripes. This phenomenon is not suppressed by the surface roughness and is general for other cell types (fibroblast and cervical carcinoma cells), too. The data are discussed with view to further application of NCD thin films in biotechnology and bio-electronics applications.

GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis.

Adsorption of fetal bovine serum on H/O-terminated diamond studied by atomic force microscopy

We investigate adsorption of fetal bovine serum (FBS), a crucial component for cell growth, on intrinsic CVD mono-crystalline diamond with hydrogen and oxygen surface terminations. The surface terminations are prepared by oxygen and hydrogen plasma process. The FBS adsorption is done by immersing diamond into McCoy's 5A medium supplemented with 15% FBS for 10 s–24 h. After rinsing the samples are characterized in the McCoy's medium and for comparison also in air by atomic force microscopy (AFM). By optimized measurements of AFM topography in oscillating regime and by using advanced AFM regimes such as phase imaging, nanoshaving, and force spectroscopy we characterize FBS thickness, adhesion, conformation and selectivity on the H/O-diamond surfaces. We find that FBS is adsorbed in about the same monolayer thickness (2–4 nm) on both H/O-diamond surfaces, yet the protein conformation is different. We present a schematic model.

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.

Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.

Myostatin Directly Regulates Skeletal Muscle Fibrosis

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.

Stage-specific expression of bone morphogenetic protein type I and type II receptor genes: Effects of follicle-stimulating hormone on ovine antral follicles

We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3–5 and >5 mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3–5 mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10 ng/ml) or a combination of 5 ng/ml FSH with 1 ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1–10 ng/ml) alone down-regulated the expression of BMPRIB ( P < 0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5 ng/ml compared to control. A further increase in FSH (10 ng/ml) down-regulated the expression of BMPRII and ALK-5 ( P < 0.05). The combination of FSH (5 ng/ml) and E2 (1 ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells ( P < 0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.

The eIF2α Kinases PERK and PKR Activate Glycogen Synthase Kinase 3 to Promote the Proteasomal Degradation of p53

Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2alpha kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2alpha kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2alpha kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2alpha kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2alpha kinases and p53 with implications in cell proliferation and tumorigenesis.