Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-α and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm2) on TNF-α and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-α or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-α and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-α activation of JNK. These findings indicate that fluid shear stress inhibits TNF-α-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-α signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a “fold-specific” inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription–PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Kinesin molecular motors: Transport pathways, receptors, and human disease

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation

The stress-activated protein kinase p38 is often induced by cytotoxic agents, but its contribution to cell death is ill defined. In Rat-1 cells, we found a strong correlation between activation of p38 and induction of c-Myc–dependent apoptosis. In cells with deregulated c-Myc expression but not in control cells, cis-diamminedichloroplatinum induced p38 activity and typical features of apoptosis, including internucleosomal DNA degradation, induction of caspase activities, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not block p38 activation and the p38 inhibitor SB203580 had no detectable effect on the activation of caspases or the in vivo cleavage of several caspase substrates, suggesting that p38 and caspase activation can contribute distinct features of apoptosis. Accordingly, we found that cell blebbing was independent of caspase activity and, rather, depended on p38-sensitive changes in microfilament dynamics likely mediated by heat shock protein 27 phosphorylation. Furthermore, p38 activity contributed to both caspase-dependent and caspase-independent nuclear condensation and fragmentation, suggesting a role in an early event triggering both mechanisms of apoptosis or sensitizing the cells to the action of both types of apoptosis executioners. Inhibiting p38 also resulted in a significant enhancement in cell survival estimated by colony formation. This capacity to modulate the sensitivity to apoptosis in cells with deregulated c-Myc expression suggests an important role for p38 in tumor cell killing by chemotherapeutic agents.

Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells.

Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways

The docking protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6.

The biological function of the retinoblastoma protein (RB) in the cell division cycle has been extensively documented, but its apparent role in differentiation remains largely unexplored. To investigate how RB is involved in differentiation, the U937 large-cell lymphoma line was induced to differentiate along a monocyte/macrophage lineage. During differentiation RB was found to interact directly through its simian virus 40 large tumor antigen (T antigen)-binding domain with NF-IL6, a member of the CAAT/enhancer-binding protein (C/EBP) family of transcription factors. NF-IL6 utilizes two distinct regions to bind to the hypophosphorylated form of RB in vitro and in cells. Wild-type but not mutant RB enhanced both binding activity of NF-IL6 to its cognate DNA sequences in vitro and promoter transactivation by NF-IL6 in cells. These findings indicate a novel biochemical function of RB: it activates, by an apparent chaperone-like activity, specific transcription factors important for differentiation. This contrasts with its sequestration and inactivation of other transcription factors, such as E2F-1, which promote progression of the cell cycle. Such disparate mechanisms may help to explain the dual role of RB in cell differentiation and the cell division cycle.

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

Osmotic regulation of cytokine synthesis in vitro.

These studies were undertaken to investigate the therapeutic mechanism of saturated solutions of KI, used to treat infectious and inflammatory diseases. The addition of 12-50 mM KI to cultured human peripheral blood mononuclear cells resulted in 319-395 mosM final solute concentration and induced interleukin (IL)-8 synthesis. Maximal IL-8 production was seen when 40 mM salt was added (375 mosM) and was equal to IL-8 induced by endotoxin or IL-1 alpha. However, there was no induction of IL-1 alpha, IL-1 beta, or tumor necrosis factor to account for the synthesis of IL-8; the effect of KI was not due to contaminating endotoxins. Hyperosmolar NaCl also induced IL-8 and increased steady-state levels of IL-8 mRNA similar to those induced by IL-1 alpha. IL-8 gene expression was elevated for 96 hr in peripheral blood mononuclear cells incubated with hyperosmolar NaCl. In human THP-1 macrophagic cells, osmotic stimulation with KI, NaI, or NaCl also induced IL-8 production. IL-1 signal transduction includes the phosphorylation of the p38 mitogen-activated protein kinase that is observed following osmotic stress. Using specific blockade of this kinase, a dose-response inhibition of hyperosmolar NaCl-induced IL-8 synthesis was observed, similar to that in cells stimulated with IL-1. Thus, these studies suggest that IL-1 and osmotic shock utilize the same mitogen-activated protein kinase for signal transduction and IL-8 synthesis.

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.