Par-4 Is an Essential Downstream Target of DAP-like Kinase (Dlk) in Dlk/Par-4–mediated Apoptosis

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A–expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.     

Estrogen Receptor (ER)-α36 Is Involved in Estrogen- and Tamoxifen-Induced Neuroprotective Effects in Ischemic Stroke Models

The neuroprotection by estrogen (E2) and tamoxifen is well documented in experimental stroke models; however, the exact mechanism is unclear. A membrane-based estrogen receptor, ER-α36, has been identified. Postmenopausal-levels of E2 act through ER-α36 to induce osteoclast apoptosis due to a prolonged activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signaling. We hypothesized that ER-α36 may play a role in the neuroprotective activities of estrogen and tamoxifen. Here, we studied ER-α36 expression in the brain, as well as its neuroprotective effects against oxygen and glucose deprivation (OGD) in PC12 cells. We found that ER-α36 was expressed in both rat and human brain. In addition, OGD-induced cell death was prevented by l nmol/L 17β-estradiol (E2β). E2β activates the MAPK/ERK signaling pathway in PC12 cells under basal and OGD conditions by interacting with ER-α36 and also induces ER-α36 expression. Low-dose of tamoxifen up-regulated ER-α36 expression and enhanced neuronal survival in an ovariectomized ischemic stroke model. Furthermore, low-dose of tamoxifen enhanced neuroprotective effects by modulating activates or suppress ER-α36. Our results thus demonstrated that ER-α36 is involved in neuroprotective activities mediated by both estrogen and tamoxifen.     

Na/K Pump and Intracellular Monovalent Cations: Novel Mechanism of Excitation–Transcription Coupling Involved in Inhibition of Apoptosis

The Na/K pump plays a key role in the regulation of the intracellular concentrations of monovalent cations and related cell function leading to electrogenesis and excitation–contraction coupling. We focus this review on the analysis of recent data showing that (i) inhibition of the Na/K pump triggers a signaling cascade independently of modulation of the intracellular [Na⁺]_i/[K⁺]_i ratio; (ii) elevation of [Na⁺]_i under sustained inhibition of the Na/K pump leads to expression of a set of genes by [Ca²⁺]_i-dependent and independent pathways; (iii) [Na⁺]_i-sensitive genes are involved in the inhibition of programmed cell death (apoptosis) in vascular smooth muscle cells.     

AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts

Background

CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts.

Results

We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14–21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion.

Conclusions

This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts.     

Novel Polymorphisms in Porcine 3′UTR of the Leptin Gene,Including a Rare Variant within Target Sequence for MIR-9 Gene in Duroc Breed,not Associated with Production Traits

The leptin gene (LEP) is considered as a functional candidate for production traits in livestock due to its crucial role in energy homeostasis. Because polymorphisms in regulatory sequences may affect gene expression, we searched for them in the 3′UTR of LEP and analyzed their association with production traits. Four breeds and a composite line were studied. In the Polish Landrace and Polish Large White breeds, 8 SNPs and 1 indel were observed; whereas, in the Duroc breed, 9 specific SNPs were found. Pietrain and Line 990 were monomorphic. One SNP (g.+168C>T), observed in the Duroc breed only, was located within a target site for microRNA (miR-9). Association studies showed a weak association between one SNP (c.+846C>T) and abdominal fat weight in the Polish Landrace only. Thus, we concluded that contribution of polymorphisms in the 3′UTR to phenotypic variability of pig production traits is marginal. Moreover, we presented an overview of known polymorphisms (128) in the pig leptin gene.     

Doxorubicin in Combination with a Small TGFβ Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models

Background

Recent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models.

Principal Findings

We report that chemotherapeutic drug doxorubicin activates TGFβ signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TβR1-KI was administered in combination with doxorubicin.

Conclusions

These observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis.     

Novel Binding of the Mitotic Regulator TPX2 (Target Protein for Xenopus Kinesin-like Protein 2) to Importin-��

Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-α. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-α, which occurs when the latter is bound to importin-β. The importin-α:β interaction is disrupted by the high RanGTP concentration near the chromosomes, releasing TPX2. In more distal regions, where Ran is predominantly GDP-bound, TPX2 remains bound to importin-α and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-α. A 2.2 Å resolution crystal structure shows that the primary nuclear localization signal (²⁸⁴KRKH²⁸⁷) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-α. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-α variants in which binding to either the major or minor NLS-binding site was impaired, together with competition assays using the SV40 monopartite NLS that binds primarily to the major site. The different way in which TPX2 binds to importin-α could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-α from other NLS-containing proteins.     

Normal Superoxide Dismutase (SOD) Gene in Pregnancy-Induced Hypertension: Is the Decreased Sod Activity a Secondary Phenomenon?

Reactive oxygen species (ROS) have been implicated in the pathogenesis of pregnancy-induced hypertension (PIH). A genetic factor is also thought to be associated with the disease. The aim of the present study was to investigate whether decreased superoxide dismutase (SOD) activity in PIH resulted from gene abnormalities. Fourteen patients with PIH were enrolled in the study. Normal pregnant women and normal nonpregnant women served as controls. Genomic DNA and mRNA were isolated from white cells and subjected to Southern and Northern blot analysis with a 600 bp CuZn-SOD probe. SOD activity was also determined in the white blood cells and red blood cells. The results showed that SOD activity was significantly reduced in patients with PIH compared to both control groups. There were no significant differences in the size of the CuZn-SOD gene and its expression between the patients with PIH and the controls. This study confirmed that there was a decreased SOD activity in PIH but revealed neither major structural changes in the genomic DNA nor mRNA size of CuZn-SOD. Our results suggest that the decreased SOD levels in PIH are not due to abnormalities in the CuZn-SOD gene and are an acquired phenomenon which occurs during the development of the disease.     

Is Contact Between Conspecifics Involved in the Cohesion of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) Aggregations?

The multicolored Asian ladybeetle, Harmonia axyridis (Pallas), exhibits a gregarious behavior during unfavorable winter conditions. Although this behavior is currently described as a phenomenon occurring only during winter, H. axyridis aggregations can also be observed outside overwintering conditions. However, the substrate markings previously highlighted as being involved in the wintry aggregation of this exotic species do not seem to be used by non-overwintering individuals to aggregate. This fact suggests then that other cues are responsible for the induction of this behavior. In this work, we have tested the hypothesis that direct contact between non-overwintering individuals stimulates the establishment of clusters. Binary choice experiments highlighted the involvement of elytral cuticular compounds in this phenomenon. Chromatographic analyses showed that the active extracts contained mainly hydrocarbons, including saturated, mono-unsaturated, and di-unsaturated homologues. Physical contact also seems to be involved in the non-overwintering aggregative behavior of H. axyridis, but to a lesser extent than these natural compounds. These findings could eventually be used to develop new control methods of these pest populations and so, reduce the adverse impacts it causes on biodiversity.     

Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-ɣ): Molecular Effects and Its Importance as a Novel Therapeutic Target for Cerebral Ischemic Injury

Neurochemical Research - Cerebral ischemic injury is a leading cause of death and long-term disability throughout the world. Peroxisome proliferator-activated receptor gamma (PPAR-?) is a...     

Designing a Novel Multi-epitope T Vaccine for “Targeting Protein for Xklp-2” (TPX2) in Hepatocellular Carcinoma Based on Immunoinformatics Approach

International Journal of Peptide Research and Therapeutics - Hepatocellular carcinoma (HCC) is one of the leading cancer-related deaths worldwide. Recently, studies for HCC treatment are focused on...     

Intracellular signalling: Is PIP(2) a messenger too?

The phospholipid phosphatidylinositol (4,5) bisphosphate (PIP(2)) has recently been shown to act downstream of the small G proteins Rac and Arf. Different effectors may be employed in each case, suggesting that PIP(2) has multiple signalling roles.     

Stain–Decolorize–Stain (SDS): a new technique for multiple staining

Multiple staining of more than one gene/antigen on a single tissue section is an indispensable tool in cell and tissue research. However, most of the available multiple staining techniques have limitations, and there has been no technique to simultaneously visualize and distinguish tissue antigens, nucleotide sequences and other chemical compounds on the same slide. Here, we present a practical and economic multiple stain technique, with which multiple cellular components including mRNA (with in situ hybridization), antigen epitope (with immunohistochemistry) and chemical molecules (with histochemistry) can be stained on a single tissue section to study their relationship. In addition, this technique also offers the possibility to evaluate morphology with an H&E staining on the same sections. We used the placenta, pancreas, breast ductal carcinoma, colon adenocarcinoma, cerebellum, tonsil and heart tissue sections to evaluate the applicability of this new technique. The sensitivity and specificity of the technique have been tested, and an optimal protocol is recommended. Its applications in surgical pathology and research are discussed. This technique offers a novel tool to evaluate the relationship among multiple components at the same or adjacent locations to meet the needs of pathology diagnosis and research.     

SMG-ly Knocking Out Gene Expression in Specific Cells: An Educational Primer for Use with “A Novel Strategy for Cell-Autonomous Gene Knockdown in Caenorhabditis elegans Defines a Cell-Specific Function for the G-Protein Subunit GOA-1”

SUMMARY

A recent article by Maher et al. in GENETICS introduces an alternative approach to cell-type-specific gene knockdown in Caenorhabditis elegans, using nonsense-mediated decay. This strategy has the potential to be applicable to other organisms (this strategy requires that animals can survive without nonsense-mediated decay—not all can). This Primer article provides a guide and resource for educators and students by describing different gene knockdown methodologies, by assisting with the technically difficult portions of the Maher et al. article, and by providing conceptual questions relating to the article.Related article in GENETICS: Maher, K. N., A. Swaminathan, P. Patel, and D. L. Chase, 2013 A novel strategy for cell-autonomous gene knockdown in Caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1. Genetics 194: 363–373.