Search

The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori. [ABSTRACT FROM AUTHOR]

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and cobrectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following; (i) substitution of GIyA1 by D-Ala or D-Leu, and (ii) substitution of ThrAS by diaminobutyric acid (Dab). The crystal structure of [D-AlaA1,DabA8]insulin, as determined within a T6 zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer. [ABSTRACT FROM AUTHOR]

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the β2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, β-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N- terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated β-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxi- dant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation. [ABSTRACT FROM AUTHOR]

Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor α1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblast differentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents. [ABSTRACT FROM AUTHOR]

The biological antagonism between the signaling proteins Numb and Notch has been implicated in the regulation of many developmental processes, especially in asymmetric cell division. Mechanistic studies show that Numb inactivates Notch via endocytosis and proteasomal degradation that directly reduce Notch protein levels at the cell surface. However, some aspects of how Numb antagonizes Notch remain unclear. Here, we report a novel mechanism in which Numb acts as a Notch antagonist by controlling the intracellular destination and stability of the Notch ligand Delta-like 4 (Dll4) through a postendocytic-sorting process. We observed that Numb/Numblike knockdown increases the stability and cell-surface accumulation of Dll4. Further study indicated that Numb acts as a sorting switch to control the postendocytic trafficking of Dll4. Of note, the Numb/Numblike knockdown decreased Dll4 delivery to the lysosome, while increasing the recycling of Dll4 to the plasma membrane. Moreover, we demonstrate that this enrichment of Dll4 at the cell surface within Numb/Numblike knockdown cells could activate Notch signaling in neighboring cells. We also provide evidence that Numb negatively controls the Dll4 plasma membrane recycling through a well-documented recycling regulator protein AP1. In conclusion, our study has uncovered a molecular mechanism whereby Numb regulates the endocytic trafficking of the Notch ligand Dll4. Our findings provide a new perspective on how Numb counteracts Notch signaling and sheds additional critical insights into the antagonistic relationship between Numb and Notch signaling. [ABSTRACT FROM AUTHOR]

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer. [ABSTRACT FROM AUTHOR]

In this study, we report that lipocalin 2 (Lcn2), a recently characterized adipokine/cytokine, is a novel regulator of brown adipose tissue (BAT) activation by modulating the adrenergic independent p38 MAPK-PGC-1α-UCP1 pathway. Global Lcn2 knock-out (Lcn2-/-) mice have defective BAT thermogenic activation caused by cold stimulation and decreased BAT activity under high fat diet-induced obesity. Nevertheless, Lcn2-/- mice maintain normal sympathetic nervous system activation as evidenced by normal catecholamine release and lipolytic activity in response to cold stimulation. Further studies showed that Lcn2 deficiency impairs peroxisomal and mitochondrial oxidation of lipids and attenuates cold-induced Pgc1a and Ucp1 expression and p38 MAPK phosphorylation in BAT. Moreover, in vitro studies showed that Lcn2 deficiency reduces the thermogenic activity of brown adipocytes. Lcn2-/- differentiated brown adipocytes have significantly decreased expression levels of brown fat markers, decreased p38 MAPK phosphorylation, and decreased mitochondrial oxidation capacity. However, Lcn2-/- brown adipocytes have normal norepinephrine-stimulated p38MAPKand hormone-sensitive lipase phosphorylation and Pgc1a and Ucp1 expression, suggesting an intact β-adrenergic signaling activation. More intriguingly, recombinant Lcn2 was able to significantly stimulate p38 MAPK phosphorylation in brown adipocytes. Activating peroxisome proliferator-activated receptor γ, a downstream effector of PGC-1α, by thiazolidinedione administration fully reverses the BAT function of Lcn2-/- mice. Our findings provide evidence for the novel role Lcn2 plays in oxidative metabolism and BAT activation via an adrenergic independent mechanism. [ABSTRACT FROM AUTHOR]

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ∼50-80% of Kv1.3 channel currents at a concentration of 1μM. The exception was rBm- KTT-3, which had weak activity. The IC50 values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ∼129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel- interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitztype potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Proliferation inhibition of vascular smooth muscle cells (VSMCs) is governed by the activity of a transcription factor network. Krüppel-like factor 4 (KIf4), retinoic acid receptor (RARα), and platelet-derived growth factor receptor (PDGFR) are expressed in VSMCs and are components of such a network. However, the relationship among them in the regulation of VSMC proliferation remains unknown. Here, we investigated the mechanisms whereby Klf4 mediates the growth inhibitory effects in VSMCs through RARα and PDGFRβ. We demonstrated that KIf4 directly binds to the 5' regulatory region of RARα, down-regulates RARn expression, and specifically inhibits RARα-mediated phosphatidylinositol 3-kinase (PI3K) and ERK signaling in cultured VSMCs induced by the synthetic retinoid Am80. Of particular interest, KIf4 inhibits RARn and PDGFRβ expression while blocking PI3K and ERK signaling induced by Am80 and PDGF-BB, respectively. The anti-proliferative effects of KIf4 on neointimal formation depend largely on PDGFR-mediated PI3K signaling without involvement of the RARα-activated signaling pathways. These findings provide a novel mechanism for signal suppression and growth inhibitory effects of KIf4 in VSMCs. Moreover, the results of this study suggest that KIf4 is one of the key mediators of retinoid actions in VSMCs. [ABSTRACT FROM AUTHOR]

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1N17) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization. [ABSTRACT FROM AUTHOR]

Although human papillomavirus (HPV) infections are the primary cause of cervical cancer, the molecular mechanism by which HPV induces cervical cancer remains largely unclear. We used two-dimensional electrophoresis with mass spectrometry to study protein expression profiling between HPV16-positive cervical mucosa epithelial H8 cells and cervical cancer Caski cells to identify 18 differentially expressed proteins. Among them, retinoblastoma-binding protein 4 (RbAp48) was selected, and its differentiation expression was verified with both additional cervical cancer-derived cell lines and human tissues of cervical intraepithelial neoplasia and cervical cancer. Suppression of RbAp48 using small interfering RNA approach in H8 cells significantly stimulated cell proliferation and colony formation and inhibited senescence-like phenotype. Remarkably, H8 cells acquired transforming activity if RpAp48 was sup- pressed, because H8 cells stably transfected with RbAp48 small interfering RNA led to tumor formation in nude mice. In addition, overexpression of RbAp48 significantly inhibited cell growth and tumor formation. This RbAp48-mediated transformation of HPV16 is probably because of the regulation by RbAp48 of tumor suppressors retinoblastoma and p53, apoptosis-related enzymes caspase-3 and caspase-8, and oncogenic genes, including E6, £7, cyclin D1 (CCND1), and c-MYC. In brief, RbAp48, previously unknown in cervical carcinogenesis, was isolated in a global screen and identified as a critical mediator controlling the transforming activity of HPV16 in cervical cancer. [ABSTRACT FROM AUTHOR]

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3-/- knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/-, neonates survived. The lungs from E18.5 and E195Abca3-/- mice were less mature than wild type. Alveolar type 2 cells from Abca3-/- embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/- compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidyicholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3-/-embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation. [ABSTRACT FROM AUTHOR]

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. [ABSTRACT FROM AUTHOR]

The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are regulated still remain to be elucidated. We describe here a novel mutine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro. Remarkably, the expression of a catalytically inactive point mutant of Wwp2 abolishes Oct-4 ubiquitination. Moreover, Wwp2 promotes Oct-4 degradation in the presence of overexpressed ubiquitin. The degradation is blocked by treatment with proteasome inhibitor. Fusion of a single ubiquitin to Oct-4 inactivates its transcriptional activity in a heterologous Oct4-driven reporter system. Furthermore, overexpression of Wwp2 in embryonic stem cells significantly reduces the Oct-4-transcriptional activities. Collectively, we demonstrate for the first time that Oct-4 can be posttranslationally modified by ubiquitination and that this modification dramatically suppresses its transcriptional activity. These results reveal that the functional status of Oct-4, in addition to its expression level, dictates its transcriptional activity, and the results open up a new avenue to understand how Oct-4 defines the fate of embryonic stem cells. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-κB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-κB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-κBα expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-κB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-κB subunits p50 and p65 bound to the NF-κB response elements of the BMP-2 gene. Thus, NF-κB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-κB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2′-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-κB, which may play an important role in chondrogenesis. [ABSTRACT FROM AUTHOR]