Dysregulation of transforming growth factor beta signaling in scleroderma: overexpression of endoglin in cutaneous scleroderma fibroblasts.

OBJECTIVE: As an initial approach to understanding the basis of the systemic sclerosis (SSc; scleroderma) phenotype, we sought to identify genes in the transforming growth factor beta (TGF beta) signaling pathway that are up-regulated in lesional SSc fibroblasts relative to their normal counterparts. METHODS: We used gene chip, differential display, fluorescence-activated cell sorter, and overexpression analyses to assess the potential role of TGF beta signaling components in fibrosis. Fibroblasts were obtained by punch biopsy from patients with diffuse cutaneous SSc of 2-14 months' duration (mean 8 months) and from age- and sex-matched healthy control subjects. RESULTS: Unexpectedly, we found that fibroblasts from SSc patients showed elevated expression of the endothelial cell-enriched TGF beta receptor endoglin. Endoglin is a member of the nonsignaling high-affinity TGF beta receptor type III family. The expression of endoglin increased with progression of disease. Transfection of endoglin in fibroblasts suppressed the TGF beta-mediated induction of connective tissue growth factor promoter activity. CONCLUSION: SSc is characterized by overproduction of matrix; that is, genes that are targets of TGF beta signaling in normal fibroblasts. Our findings suggest that lesional SSc fibroblasts may overexpress endoglin as a negative feedback mechanism in an attempt to block further induction of profibrotic genes by TGF beta.

Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells.

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.

In vivo association of Grb2 with pp116, a substrate of the T cell antigen receptor-activated protein tyrosine kinase.

Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.

Beta 1-adrenergic and dopamine (D1)-receptors coupled to adenylyl cyclase activation in GT1 gonadotropin-releasing hormone neurosecretory cells.

Binding sites labeled by the beta-adrenergic receptor radioligand (-)-[125I]iodocyanopindolol ([125I]ICYP) and the selective D1-subtype dopamine (DA) receptor radioligand (+)-[125I]SCH 23982 were identified on immortalized hypothalamic GnRH neurons (GT1-7 cell lines). Saturation analyses in crude particulate suspensions of GT1 cells described high affinity and low capacity binding sites for [125I]ICYP (Kd, 41 pM; binding capacity, 25 fmol/mg protein) and [125I]SCH 23982 (Kd, 320 pM; binding capacity, 23 fmol/mg protein). These binding sites were further characterized in competition assays using a variety of agonists and antagonists selective for either beta-adrenergic or DA receptor subtypes. The pharmacological profiles of [125I]ICYP and [125I]SCH 23982 binding obtained from these studies indicated that the radioligands were labeling beta 1-adrenergic and D1-dopaminergic receptor sites, respectively. Northern blot analyses of purified GT1 cell mRNA documented the expression of D1-dopaminergic and beta 1-adrenergic receptor mRNAs. beta 2-Adrenergic receptor mRNA was not identified. All three transcripts were detected in mouse brain mRNA. Both beta 1-adrenergic and D1-receptors were discovered to be positively coupled to adenylyl cyclase. DA and the beta-adrenergic agonist isoproterenol each provoked a rapid and marked stimulation of adenylyl cyclase activity in GT1 cell membrane suspensions. Subtype-selective beta-adrenergic and DA receptor antagonists were used to inhibit isoproterenol- and DA-stimulated adenylyl cyclase activities. Their relative potencies indicated that the isoproterenol stimulation was mediated via the beta 1-adrenergic receptor. The DA-stimulated adenylyl cyclase activity was mediated via the D1-DA receptor. These studies have identified functional beta 1-adrenergic and D1-dopaminergic receptors positively coupled to adenylyl cyclase on GT1 GnRH neurosecretory cells. The existence of these receptors suggests that the noradrenergic and dopaminergic regulation of gonadotropin secretion may be mediated at least in part via direct synapses on GnRH neurons.

Automated stability-indicating high-performance liquid chromatographic assay for ethinyl estradiol and (levo)norgestrel tablets.

An automated high-performance liquid chromatography (HPLC) assay for ethinyl estradiol and norgestrel or levonorgestrel in oral contraceptive tablets was developed. Tablets were prepared for on-line injection using a solid sampler and segmented continuous flow techniques. The active components were separated from tablet excipients, impurities, and degradation products on reversed-phase C8 and C18 columns by elution with water-acetonitrile-methanol (45:35:15). A UV detector connected in series with a fluorometric detector measured the UV absorbance of levonorgestrel and norgestrel at 240 nm and the fluorescence of ethinyl estradiol at 310 nm (excitation at 210 nm). The method employed computer control of the injection system and solid sampler for synchronization of the chromatographic and segmented flow streams. The method is applicable for content uniformity and stability testing at a rate of eight samples per hour.