The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4+ T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell–cell fusion, and examined levels of SDF-1 transcripts in CD8+ T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the α and β forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101–1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein

Biological sensing of small molecules such as NO, O2, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein’s response to CO is proposed.

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: A novel mechanism of growth hormone receptor regulation

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 ± 2.9% and 23.0 ± 2.7% of control (mean ± SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P ≤ 0.0005), whereas GHR translocation was completely abolished by 75–90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (XHX), and FVIII-deficient (XHY and XHXH) fetuses injected with adenoviral vectors carrying luciferase or β-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 × 105 plaque-forming units) was 87%. FVIII activity in plasma was 50.7 ± 10.5% of normal levels at day 2 of life, 7.2 ± 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.

Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (fgf-as) from neonatal rat liver. In non-central nervous system (CNS) tissues the fgf-as RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and fgf-as RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.

Regulation of Bcl-xL expression in human keratinocytes by cell–substratum adhesion and the epidermal growth factor receptor

Cell–substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell–substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.