The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a "switch" type regulator. We identified through immunoprecipitation-mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Ovarian Neoplasms ; Phosphatidylinositol 3-Kinases ; Proteomics ; Repressor Proteins ; TOR Serine-Threonine Kinases ; Tacrolimus Binding Proteins

Paget's disease of bone (PDB) is a common metabolic bone disease that is characterized by aberrant focal bone remodeling, which is caused by excessive osteoclastic bone resorption followed by disorganized osteoblastic bone formation. Genetic factors are a critical determinant of PDB pathogenesis, and several susceptibility genes and loci have been reported, including SQSTM1, TNFSF11A, TNFRSF11B, VCP, OPTN, CSF1 and DCSTAMP. Herein, we report a case of Chinese familial PDB without mutations in known genes and identify a novel c.163G>C (p.Val55Leu) mutation in FKBP5 (encodes FK506-binding protein 51, FKBP51) associated with PDB using whole-exome sequencing. Mutant FKBP51 enhanced the Akt phosphorylation and kinase activity in cells. A study of osteoclast function using FKBP51V55L KI transgenic mice proved that osteoclast precursors from FKBP51V55L mice were hyperresponsive to RANKL, and osteoclasts derived from FKBP51V55L mice displayed more intensive bone resorbing activity than did FKBP51WT controls. The osteoclast-specific molecules tartrate-resistant acid phosphatase, osteoclast-associated receptor and transcription factor NFATC1 were increased in bone marrow-derived monocyte/macrophage cells (BMMs) from FKBP51V55L mice during osteoclast differentiation. However, c-fos expression showed no significant difference in the wild-type and mutant groups. Akt phosphorylation in FKBP51V55L BMMs was elevated in response to RANKL. In contrast, IκB degradation, ERK phosphorylation and LC3II expression showed no difference in wild-type and mutant BMMs. Micro-CT analysis revealed an intensive trabecular bone resorption pattern in FKBP51V55L mice, and suspicious osteolytic bone lesions were noted in three-dimensional reconstruction of distal femurs from mutant mice. These results demonstrate that the mutant FKBP51V55L promotes osteoclastogenesis and function, which could subsequently participate in PDB development.
Acid Phosphatase ; Animals ; Asian Continental Ancestry Group ; Bone Diseases, Metabolic ; Bone Remodeling ; Bone Resorption ; Femur ; Humans ; Mice ; Mice, Transgenic ; Osteitis Deformans* ; Osteoblasts ; Osteoclasts ; Osteogenesis ; Phosphorylation ; Phosphotransferases ; Tacrolimus Binding Proteins ; Transcription Factors

Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.
Active Transport, Cell Nucleus/drug effects ; *Apoptosis ; Aspirin/*pharmacology ; Cell Nucleus/*metabolism ; Humans ; MCF-7 Cells ; Phosphorylation ; Protein Binding ; Proto-Oncogene Proteins c-bcl-2/genetics/*metabolism ; Tacrolimus Binding Proteins/metabolism

OBJECTIVETo obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses.

METHODSThe N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF.

RESULTSThe recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells.

CONCLUSIONWe successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.

Antibodies, Monoclonal ; biosynthesis ; Fluorescent Antibody Technique ; Genetic Vectors ; genetics ; Humans ; Immunohistochemistry ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Tacrolimus Binding Proteins ; biosynthesis ; genetics ; immunology

significant advances have been made in understanding the biological underpinnings of post-traumatic stress disorder(PTSD), particularly in the field of genetics and neuroimaging. Association studies in candidate genes related with hypothalamic-pituitary-adrenal axis, monoamines including serotonin, dopamine and noradrenaline, and proteins including FK506-binding protein 5 and brain-derived neurotrophic factor have provided important insights with regard to the vulnerability factors in PTSD. Genome-wide association studies and epigenetic studies may provide further information for the role of genes in the pathophysiology of PTSD. Hippocampus, medial prefrontal cortex, anterior cingulated cortex and amygdala have been considered as key structures that underlie PTSD pathophysiology. Future research that combines genetic and neuroimaging information may provide an opportunity for a more comprehensive understanding of PTSD.
Amygdala ; Axis, Cervical Vertebra ; Brain-Derived Neurotrophic Factor ; Dopamine ; Epigenomics ; Genome-Wide Association Study ; Hippocampus ; Neuroimaging ; Norepinephrine ; Prefrontal Cortex ; Proteins ; Serotonin ; Stress Disorders, Post-Traumatic ; Tacrolimus Binding Proteins

PURPOSE: FK506 (tacrolimus) is a widely used immunosuppressive agent in the treatment of various medical conditions, including autoimmune disease, bone marrow and organ transplantations. Previously FK506 was known to cause apoptotic death of human Jurkat T cells. METHODS: The current study was designed to analyze the gene expression pattern of Jurkat T cells after FK506 application by using cDNA microarray. Treatment of Jurkat T cells with FK506 resulted in a decrease of cell viability in a time- and dose-dependent manner. Next, total RNA of Jurkat T cells was extracted by using TRIzol reagent and used to carry out a confirmation test for the purity and integrity of total RNA. RESULTS: Gene expression levels related to apoptosis and cell cycle process were mainly focused to analyze in FK506-treated Jurkat T cells. According to the inhibition of calcineurin activity, MARCKS in PKC substrates and Sp3 transcription factor was markedly increased in FK506-treated cells. Also, cell cycle control gene Id1 and Id3 were induced in expression from FK506-treated Jurkat T cells. However, FK506 decreased the expression of Src homology 2, G protein, and MEK 2 genes in bioactive peptide induced signaling pathway. It also reduced the expression level of the insulin receptor, DRPLA and Bai1-associated protein 2 genes, which are involved in the regulation of cell motility and morphology control. CONCLUSION: The author will continue to pursue the exact functional roles of genes that are markedly changed in expression by FK506 in human Jurkat T cells in vitro and in vivo experimental models.
Apoptosis ; Autoimmune Diseases ; Bone Marrow ; Calcineurin ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Movement ; Cell Survival ; Gene Expression ; Gene Expression Profiling ; GTP-Binding Proteins ; Guanidines ; Humans ; Jurkat Cells ; Models, Theoretical ; Oligonucleotide Array Sequence Analysis ; Organ Transplantation ; Phenols ; Receptor, Insulin ; RNA ; Sp3 Transcription Factor ; T-Lymphocytes ; Tacrolimus ; Transplants

OBJECTIVETo investigate 2 polymorphism sites in exon 3 and intron 2 of FKBP6 in Chinese population, while screening the gene mutations and polymorphisms in exons 3, 4 of FKBP6, and the association of these polymorphisms with azoospermia.

METHODSPossible variations of exons 3, 4 and genotypes and frequencies of 2 polymorphic loci were examined by denaturing high-performance liquid chromatography(DHPLC) and PCR-restriction fragment length polymorphism(PCR-RFLP) technique in 177 azoospermia patients and 231 control individuals.

RESULTSThe observed allele frequencies conformed well to Hardy-Weinberg equilibrium. The frequency of 278A allele was significantly higher in controls than that in patients (P<0.05). C/T(s7797242) polymorphism was not found in either group and variations in exons 3, 4 were not detected.

CONCLUSION278A polymorphism of FKBP6 gene was associated with idiopathic azoospermia, while C/T, 370G/A, 430G/C, 467T/C, 468G/A polymorphisms might be very rare in Chinese population.

Azoospermia ; genetics ; Chromatography, High Pressure Liquid ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Tacrolimus Binding Proteins ; genetics

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.
1-Phosphatidylinositol 3-Kinase/metabolism ; Drug Synergism ; Enzyme Activation ; Granulocyte-Macrophage Colony-Stimulating Factor/*pharmacology ; Hematopoietic Stem Cells/*enzymology ; Human ; Phosphorylation/drug effects ; Protein-Serine-Threonine Kinases/metabolism ; Ribosomal Protein S6 Kinases, 70kD/antagonists & inhibitors/*metabolism ; Stem Cell Factor/*pharmacology ; Tacrolimus Binding Protein 1A/metabolism

Urolithiasis and calcium oxalate crystal deposition diseases are still significant medical problems. In the course of nephrocalcin cDNA cloning, we have identified FKBP-12 as an inhibitory molecule of calcium oxalate crystal growth. lambdagt 11 cDNA libraries were constructed from renal carcinoma tissues and screened for nephrocalcin cDNA clones using anti-nephrocalcin antibody as a probe. Clones expressing recombinant proteins, which appeared to be antigenically cross-reactive to nephrocalcin, were isolated and their DNA sequences and inhibitory activities on the calcium oxalate crystal growth were determined. One of the clone lambdagt 11 #31-1 had a partial fragment (80 bp) of FKBP-12 cDNA as an insert. Therefore, a full-length FKBP-12 cDNA was PCR-cloned from the lambdagt 11 renal carcinoma cDNA library and was subcloned into an expression vector. The resultant recombinant FKBP-12 exhibited an inhibitory activity on the calcium oxalate crystal growth (Kd=10(-7) M). Physiological effect of the extracellular FKBP-12 was investigated in terms of macrophage activation and proinflammatory cytokine gene induction. Extracellular FKBP-12 failed to activate macrophages even at high concentrations. FKBP-12 seems an anti-stone molecule for the oxalate crystal deposition disease and recurrent stone diseases.
Animals ; Base Sequence ; Calcium Oxalate/*antagonists & inhibitors ; Carcinoma, Renal Cell ; Crystallization ; DNA, Complementary ; Extracellular Space ; Glycoproteins/genetics ; Humans ; Kidney Calculi/*prevention & control ; Kidney Neoplasms ; Male ; Mice ; Mice, Inbred ICR ; Molecular Sequence Data ; Recombinant Fusion Proteins/genetics/metabolism ; Tacrolimus Binding Protein 1A/genetics/*metabolism