Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Reduced surface expression of transforming growth factor beta receptor type II in mitogen-activated T cells from Sézary patients.

Sézary syndrome (SzS), the leukemic form of cutaneous T-cell lymphoma, is characterized by clonal proliferation of CD4+ T cells and immune dysfunctions, raising the possibility of cytokine-related abnormalities. We previously described a decreased response to the growth-inhibitory effects of transforming growth factor type beta (TGF-beta) in SzS T cells accompanied by apparent loss of surface type II TGF-beta receptor (TGF beta RII). To specifically determine if defects exist in TGF beta RII protein expression and/or transport in SzS patients, we developed a sensitive flow cytometric method to detect TGF beta RII on the surface and intracellularly in the CD4+ T cells. Our results indicate that unlike normal CD4+ T cells, CD4+ T cells from 9 of 12 SzS patients expressed little, if any, surface TGF beta RII in response to mitogen stimulation. At the intracellular level, however, pools of TGF beta RII were comparable to those in normal CD4+ T cells. This indicates that defective trafficking of this inhibitory cytokine receptor may contribute significantly to the development of this disease.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor.

Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.