4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) promote adipogenesis in 3T3-L1 adipocyte cell culture.

4,4'-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4'-dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 adipocytes. Treatment of DDT or DDE resulted in increased lipid accumulation accompanied by increased expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome-proliferator activated receptor-γ (PPARγ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase, and leptin. Moreover, treatment of DDT or DDE increased protein levels of C/EBPα, PPARγ, AMP-activated protein kinase-α (AMPKα), and ACC, while significant decrease of phosphorylated forms of AMPKα and ACC were observed. These finding suggest that increased lipid accumulation caused by DDT and DDE may mediate AMPKα pathway in 3T3-L1 adipocytes.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

Vascular injury induces expression of periostin: implications for vascular cell differentiation and migration.

OBJECTIVE: Periostin mRNA is among the most strongly upregulated transcripts in rat carotid arteries after balloon injury. The goal of the present study was to gain insight into the significance of periostin in the vasculature. METHODS AND RESULTS: Periostin expression after injury was localized to smooth muscle cells of the neointima and the adventitia. The expression of periostin in smooth muscle cells in vitro was not regulated by cytokines such as fibroblast growth factor-2 (FGF-2). In contrast, stimulation of MC3T3-E1 osteoblastic cells, NIH3T3 fibroblasts, or mesenchymal C3H10T1/2 cells with FGF-2 reduced periostin mRNA levels to <5% of controls, whereas conversely bone morphogenetic protein-2 (BMP-2) increased periostin mRNA levels. Periostin expression was induced and maintained during retinoic acid-induced smooth muscle cell differentiation in A404 cells. In addition, overexpression of periostin in C3H10T1/2 cells caused an increase in cell migration that could be blocked with an anti-periostin antibody. CONCLUSIONS: Periostin expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Unlike other mesenchymally derived cell lines, periostin expression is not regulated by FGF-2 in smooth muscle cells. This distinction may be useful in discriminating smooth muscle and fibroblast lineages.

Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells.

A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP ( approximately 0.01 pmol). A linear response has been established over a range of 5-1000 pg ( approximately 0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125+/-0.010 pmol/10(6)cells) and GGPP (0.145+/-0.008 pmol/10(6)cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.

Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin.

The epithelial-mesenchymal interactions between keratinocyte stem cells and dermal papilla (DP) cells are crucial for normal development of the hair follicle as well as during hair cycling. During the cyclical regrowth of a new lower follicle, the multipotent hair follicle stem cells are stimulated to proliferate and differentiate through interactions with the underlying mesenchymal DP cells. To characterize the events occurring during the process of epithelial stem cell fate determination, we utilized a coculture system by incubating human hair follicle keratinocyte stem cells with DP cells. Using GeneChip microarrays, we analyzed changes in gene expression within the stem cells upon coculture with the DP over a 5-day time course. A number of important signaling pathways and growth factors were regulated. The hair-specific keratin 6hf (K6hf) gene proved a particularly good marker of hair differentiation, with a 7.9-fold increase in mRNA and resulting increased protein levels. The high expression of K6hf was unique to DP-induced keratinocyte differentiation, since expression of K6hf was not induced by high calcium. Since the beta-catenin signaling pathway has been implicated in hair follicle development, we examined the role of beta-catenin in our system and demonstrated that beta-catenin/lef-1 signaling is required for DP-induced hair differentiation.

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein.

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.

Small increases in pH enhance retroviral vector transduction efficiency of NIH-3T3 cells.

Increases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)- and protamine sulfate (PS)-aided retroviral transduction of NIH-3T3 cells in a serum-lot-dependent manner. The increase in Pb-aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS-aided transduction was less effective on an absolute basis than Pb-aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS-aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb-aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.

A role for Grb2-associated binder-1 in growth hormone signaling.

GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.

Mutant Rab24 GTPase is targeted to nuclear inclusions.

BACKGROUND: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity. RESULTS: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24. CONCLUSION: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

Osteoblast intracellular localization of Nmp4 proteins.

Nmp4 proteins are transcription factors that contribute to the expression of type I collagen and many of the matrix metalloproteinase genes. Numerous Nmp4 isoforms have been identified. These proteins, all derived from a single gene, have from five to eight Cys(2)His(2) zinc fingers, the arrangement of which directs specific isoforms to nuclear matrix subdomains. Nmp4 isoforms also have an SH3 binding domain, typical of cytoplasmic docking proteins. Although recent evidence indicates that Nmp4 proteins also reside in the osteoblast cytoplasm, whether they localize to specific organelles or structures is not well defined. The intracellular localization of a protein is a determinant of its function and provides insights into its mechanism of action. As a first step toward determining the functional relationship between the cytoplasmic and nuclear Nmp4 compartments, we mapped their location in the osteoblast cytoplasm. Immunocytochemical analysis of osteoblasts demonstrated that Nmp4 antibodies labeled the mitochondria, colocalized with Golgi protein 58K, and lightly stained the cytoplasm. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these proteins were specific to defined intracellular domains. Consistent with the western analyses, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected previously uncharacterized Nmp4 isoforms. These data necessarily enlarge the known Nmp4 family from nuclear matrix transcription factors to a more widely extended class of intracellular proteins.

High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation.

We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.

Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies.

BACKGROUND: Reliable assessment of cell death is now pivotal to many research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). METHODS: Cell death was induced by a variety of apoptotic stimuli in various EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluorescence microplate reader. RESULTS: Cells expressing EGFP exhibited a decrease in fluorescence when treated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, propidium iodide incorporation, or reactive oxygen species production. We also show that the decrease in EGFP fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation of the protein. CONCLUSIONS: This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS setups, making it an assay of choice for cytotoxicity evaluation.

Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

A retroviral gene trap containing a human CD2 cell surface antigen/neomycin-phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (Neo(R)) clones was obtained; 86% also expressed the CD2 cell surface antigen. Molecular analysis of a random sample of Neo(R) clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for secreted and transmembrane proteins. In each case, the signal sequence of the endogenous gene was fused in-frame to the CD2/neomycin-phosphotransferase reporter gene due to a cryptic splice acceptor site embedded in the coding region of the CD2 cDNA. When the library was transformed by IGF-1 and selected against CD2 expression, integrations were obtained in genes that are repressed by transformation. Molecular analysis of six randomly chosen integrations revealed that, in each case, U3Ceo captured a signal sequence from proteins involved in oncogenic transformation and metastatic spread.

Small-sample total RNA purification: laser capture microdissection and cultured cell applications.

Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.

Metabolic effects of dental resin components in vitro detected by NMR spectroscopy.

Earlier studies have shown that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) and the photostabilizer 2-hydroxy-4-methoxybenzophenone (HMBP) are cytotoxic and inhibit cell growth. It was the aim of this study to elucidate the underlying metabolic effects of TEGDMA and HMBP on immortal contact-inhibited Swiss albino mouse embryo cells (3T3 fibroblasts) by nuclear magnetic resonance (NMR) spectroscopy. Cell extracts and culture media were analyzed by NMR spectroscopy for metabolic changes after incubation for 24 hours with ED20-concentrations of TEGDMA and HMBP. TEGDMA could be detected in all fractions (cytosol, lipid fractions, and culture media) of 3T3 cells, while HMBP was found only in the lipid fraction accumulated at a maximum rate (51 nmol/mg DNA) compared with TEGDMA (27 nmol/mg DNA). TEGDMA increased the concentration of phosphomonoesters to 180+/-36% and decreased the phosphodiesters to 65+/-5% of controls (control = 100%). Thus, the turnover of phospholipids was enhanced, whereas content and composition of phospholipids of membranes did not alter markedly. Additionally, TEGDMA changed the metabolic state of cells, indicated by slight decreases of nucleoside triphosphates and an increase in the ratio of nucleoside diphosphates to nucleoside triphosphates, while HMBP had no effect. The most remarkable effect of TEGDMA was a nearly complete decline of the intracellular glutathione levels. Analysis of our data shows that NMR spectroscopy of cell-material interactions may reveal metabolic effects of organic test substances which are not detectable by standard in vitro assays. The comonomer TEGDMA affected the metabolism of the cells on different levels, while HMBP accumulated in the lipid fraction and induced significantly fewer effects on cell metabolism.

Imaging of dynamic changes of the actin cytoskeleton in microextensions of live NIH3T3 cells with a GFP fusion of the F-actin binding domain of moesin.

BACKGROUND: The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin. RESULTS: C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent. CONCLUSIONS: Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.

Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of DeltaF508-CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.

Increased resistance to apoptosis in cells overexpressing thymosin beta four: A role for focal adhesion kinase pp125FAK.

Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNF alpha). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp125FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tbeta4 overexpressing cells have approximately 1.4x more pp125FAK than the controls, and the kinase is approximately 2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp125FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp125FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp125FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.