The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.     

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol-resistant human breast cancer cell line MDA-MB-231

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and β-tubulins (more specifically the βIII and βIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.     

Inhibition of MAPK by prolactin signaling through the short form of its receptor in the ovary and decidua: involvement of a novel phosphatase

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR(-/-)RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR(-/-)RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR(-/-)RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor α and interleukin 1β detected with a three-color immunofluorescence technique

 Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In the present study, the kinetics of both intracellular and extracellular accumulation of TNFα and IL-1β in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNFα in monocytes starts shortly after initiation of the culture; i.e., TNFα is detectable after 1 h, reaching a peak level after 3–4 hours with 50–65% of monocytes staining positive. In parallel with its increased intracellular presence, TNFα was also found in the culture supernatant. The intracellular accumulation of IL-1β in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1β could be detected in the culture supernatant. TNFα and IL-1β can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNFα and IL-1β are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described. Accepted: 27 August 1996     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Regulation of axis determinacy by the Arabidopsis PINHEAD gene

Plants produce proximal-distal growth axes with two types of growth potential: they can be indeterminate, in which case growth continues indefinitely, or they can be determinate, in which case growth is limited to the production of a single organ or a discrete set of organs. The indeterminate shoot axes of Arabidopsis pinhead/zwille mutants frequently are transformed to a determinate state. PINHEAD (PNH) is expressed in the central domain of the developing plant: the provascular tissue, the shoot apical meristem, and the adaxial (upper) sides of lateral organ primordia. Here, we show that ectopic expression of PNH on the abaxial (lower) sides of lateral organs results in upward curling of leaf blades. This phenotype correlates with a loss of cell number coordination between the two surfaces of the blade, indicating that ectopic PNH can cause changes in cell division rates. More strikingly, moving PNH expression from the central to the peripheral domain of the embryo causes transformation of the determinate cotyledon axis to an indeterminate state. We propose that growth axes are specified as determinate versus indeterminate in a PNH-mediated step. Our results add to a growing body of evidence that radial positional information is important in meristem formation. These results also indicate that genes regulating cell division and axis determinacy are likely to be among PNH targets.     

Some neuronal cell populations express human dopamine beta-hydroxylase-lacZ transgenes transiently during embryonic development.

The 5' flanking region from the human dopamine beta-hydroxylase gene directs expression of bacterial beta-galactosidase reporter genes to a subset of adult neurons and adrenal chromaffin cells of transgenic mice. In this paper, we examine the spatial and temporal patterns of expression of these transgenes during embryogenesis. Expression begins at embryonic day 9 in the developing central and peripheral nervous systems and persists in cell populations in which expression is observed in adult transgenic mice. However, transient embryonic expression occurs in presumptive neuroblasts in developing sensory ganglia and ventrolateral neural tube that are destined to synthesize neurotransmitters other than catecholamines. These observations support the concept that some cells fated to become "non-catecholaminergic" neurons exhibit transient catecholaminergic features during their differentiation.     

Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.