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PubMed1994

Direct in vivo assessment of parathyroid hormone-calcium relationship curve in renal patients.

Secondary hyperparathyroidism (SHP) is a well documented finding even in the early stages of chronic renal failure (CRF). A sigmoidal relationship, fitting a four parameter model, links PTH secretion rate and calcium concentration changes. To our knowledge, PTH secretory parameters have only been studied in uremic patients who are in dialysis treatment. As a result of these studies, a possible role for derangement in setpoint values (that is, the serum calcium concentration corresponding to the mid-range value on the sigmoidal curve) has been suggested in the pathogenesis of SHP in CRF. Our study was undertaken to gain insight into the calcium-PTH relationship curve in the first course of CRF and to assess whether a change in any of the secretory parameters is related to the beginning of SHP. We studied 27 male renal patients with a variable degree of renal function (creatinine clearance 12 to 164 ml/min) and 9 control subjects. In all patients and controls the following parameters were evaluated: (1) basal 1,25(OH)2 vitamin D, 25(OH)vitamin D, calcitonin (CT), intact PTH; (2) GFR by Cr51EDTA clearance; (3) the sigmoidal PTH-ionized calcium relation curve, by means of a hypocalcemic stimulating test (Na2-EDTA 37 mg/kg body weight/2 hr) and a hypercalcemic test (Ca gluconate giving 8 mg/kg of body weight/2 hr of Ca element), performed on two consecutive days.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2006

Insufficiency of PTH action on bone in uremia.

Abnormal bone turnover and mineral metabolism is observed in patients on dialysis. Secondary hyperparathyroidism (SHP) develops in response to mineral metabolism changes accompanying renal failure. As a factor of disease progression, the phenomenon of skeletal resistance to parathyroid hormone (PTH) is observed. With recent advances in the treatment of SHP, over-secretion of PTH can now be controlled. However, blood PTH levels 2 to 3 times higher than normal are considered necessary to maintain normal bone turnover in patients with renal failure. Various causes of skeletal resistance to PTH have been reported, including decrease in PTH receptor in osteoblasts, accumulation of 7-84 PTH fragment, and accumulation of osteoprotegerin. This skeletal resistance to PTH is not only a high-turnover bone accompanying SHP, but may also play a crucial role in the onset of low-turnover bone disease. We have produced a rat model of renal failure with normal level of PTH secretion and analyzed the bone of this model. Our results confirmed that bone turnover is lowered accompanying renal function impairment. We also found that this lowered bone turnover is improved by intermittent administration of PTH. In addition, PTH receptor gene expression is also decreased in low-turnover bone, as is observed in high-turnover bone disease. These findings confirm the presence of skeletal resistance to PTH in low-turnover bone accompanying renal failure. Control of calcium, phosphorus, and PTH levels with the target to maintain normal bone turnover is important in maintaining the quality of life of patients on dialysis.

Animals↗
PubMed2001

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2.

INTRODUCTION: Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses. MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5. CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.

Animals↗
PubMed2005

The effect of semantic representation on toddlers' word retrieval.

PURPOSE: This study tested the hypothesis that depth of semantic representation influences toddlers' word retrieval. METHOD: Nineteen toddlers participated under 3 word learning conditions in this longitudinal study. Gestures cued attention to object shape (SHP) or function (FNC) in the experimental conditions. No semantic cue was provided under a control condition (CTL). Word learning conditions occurred on each of 3 days. On the 4th day, word retrieval was assessed across 3 levels of scaffolding (uncued picture naming, cued picture naming, picture recognition). Evidence of semantic representation was provided at fast and slow mapping intervals. RESULTS: Less scaffolding was necessary for word retrieval (uncued and cued naming) under experimental conditions than under the CTL condition. However, more SHP than FNC condition targets were retrieved for uncued picture naming. This latter difference may be related to the superior fast mapping of targets under the SHP condition. Toddlers stated object functions (slow mapping) comparably in the experimental conditions, but this was superior to CTL condition performance. CONCLUSIONS: Word retrieval is a continuous behavior that is positively influenced by semantic representation. Semantic knowledge of objects can be enriched by shape or function gestures, thereby improving toddlers' object word productions. Shape cues appear to be more effective for this purpose.

Analysis of Variance↗
PubMed1999

Dynamic tests of parathyroid hormone secretion using hemodialysis and calcium infusion cannot be compared.

BACKGROUND: Extracellular Ca++ concentration [Ca++] and parathormone (PTH) are related by a sigmoidal function. The set point of the control system is the [Ca++] that produces a half-maximal inhibition of PTH secretion. Whether or not this set point is abnormal in patients with chronic renal failure (CRF) and secondary hyperparathyroidism (SHP) is controversial. METHODS: We investigated whether the way [Ca++] is varied [hemodialysis (HD) or calcium gluconate/sodium citrate infusions (INF)] and the way the curve is constructed (four-parameter model or adapted four-parameter, created by Felsenfeld) could influence this set point. We performed dynamic tests of PTH secretion in 12 patients with CRF and SHP during either HD or INF. Both the four-parameter model or adapted four-parameter methods were used, creating four combinations: (a) hypocalcemia and hypercalcemia induced during HD, calculated by Brown's formula (HDB); (b) hypocalcemia and hypercalcemia induced during HD, calculated by Felsenfeld's formula (HDF); (c) hypocalcemia and hypercalcemia induced during infusion, calculated by Brown's formula (INFB); and (d) hypocalcemia and hypercalcemia induced during infusion, calculated by Felsenfeld's formula (INFF). RESULTS: The set points obtained with HDB correlated perfectly with those obtained with HDF (R2 = 0.999). A similar relationship was found between INFB and INFF (R2 = 0.9997). In contrast, there was no correlation between either HDB and INFB (R2 = 0.0157) or HDF and INFF (R2 = 0.0204). CONCLUSIONS: These findings indicate that the calculated [Ca++] set point in patients with CRF and SHP is determined by the way [Ca++] is varied, rather than by the mathematical model used to generate the curves. Further studies are needed to determine the differing physiological mechanisms triggered by HD and INF and the way they influence [Ca++] homeostasis in this setting.

Adolescent↗
PubMed2003

Hepatocyte nuclear factor 1 alpha: a key mediator of the effect of bile acids on gene expression.

Bile acids regulate the expression of genes involved in cholesterol homeostasis. They are ligands of the farnesoid X receptor, which induces small heterodimer partner (SHP)-1, a transcriptional repressor of bile acid synthetic enzymes. In cholestatic liver disease, hepatic bile acid concentrations are elevated and expression of the major Na+-independent bile acid uptake system, organic anion transporting polypeptide (OATP)-C (solute carrier gene family SLC21A6), is markedly decreased. Because the OATP-C gene is transcriptionally dependent on the hepatocyte nuclear factor (HNF) 1 alpha, we hypothesized that bile acids decrease OATP-C expression through direct repression of HNF1 alpha. To test this hypothesis, we studied the regulation of the human HNF1 alpha gene by bile acids. HNF1 alpha expression in cultured hepatoma cells was decreased approximately 50% after 12 hours' exposure to 100 micromol/L chenodeoxycholic acid (CDCA). Characterization of the human HNF1 alpha gene promoter identified a consensus bile acid response element that binds and is activated by HNF4 alpha. Mutagenesis of the HNF4 alpha site abolished baseline HNF1 alpha promoter activity. The central mechanism by which bile acids repress HNF1 alpha is decreased activation by HNF4 alpha. SHP directly inhibits HNF4 alpha-mediated transactivation of the HNF1 alpha promoter in cotransfection assays. In addition, HNF4 alpha nuclear binding activity is decreased by CDCA and the human HNF4 alpha gene promoter is repressed by CDCA through an SHP-independent mechanism. In conclusion, we show that repression of HNF1 alpha is an important new mechanism by which bile acids regulate the expression of HNF1 alpha-dependent genes in man. This explains the suppressive effect of bile acids on the OATP-C gene promoter, leading to decreased expression in cholestatic liver disease.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1997

Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein.

CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.

Abatacept↗
PubMed1998

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Hyperglycemia enhances angiotensin II-induced janus-activated kinase/STAT signaling in vascular smooth muscle cells.

We have shown previously that angiotensin II (Ang II) activates the janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in vascular smooth muscle cells (VSMCs) and that activation of the JAK/STAT pathway is required for Ang II induction of VSMC proliferation. In the present study, we examined the effects of hyperglycemia (HG) on Ang II-induced JAK/STAT signaling events in cultured VSMCs. HG increases Ang II-induced JAK2 tyrosine phosphorylation and promotes a partial tyrosine phosphorylation of the enzyme under basal conditions. In addition, HG increases both basal and Ang II-induced complex formation of JAK2 with the Ang II AT(1) receptor. The extent of STAT1 and STAT3 tyrosine and serine phosphorylation are also increased under HG conditions. Furthermore, the tyrosine phosphorylation and activities of the SHP-1 and SHP-2 tyrosine phosphatases, enzymes that regulate Ang II-induced JAK2 tyrosine phosphorylation, are altered by HG. SHP-1, which is responsible for JAK2 tyrosine dephosphorylation in VSMC, is completely deactivated in HG, resulting in a prolonged duration of JAK2 phosphorylation under HG conditions. HG also enhances Ang II induction of VSMC proliferation. Taken together, these data suggest that HG augments Ang II induction of VSMC proliferation by increasing signal transduction through the JAK/STAT pathway.

Angiotensin II↗
PubMed2000

Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis.

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

Acids↗
PubMed2000

Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.

Binding Sites↗
PubMed2000

The docking molecule gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common gamma chain-using cytokines.

Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells.

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.

Androstadienes↗
PubMed2001

Molecular events associated with CD4-mediated Down-regulation of LFA-1-dependent adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.

CD4 Antigens↗
PubMed2002

Regulation of the farnesoid X receptor (FXR) by bile acid flux in rabbits.

We investigated the roles of hydrophobic deoxycholic acid (DCA) and hydrophilic ursocholic acid (UCA) in the regulation of the orphan nuclear farnesoid X receptor (FXR) in vivo. Rabbits with bile fistula drainage (removal of the endogenous bile acid pool), rabbits with bile fistula drainage and replacement with either DCA or UCA, and intact rabbits fed 0.5% cholic acid (CA) (enlarged endogenous bile acid pool) were studied. After bile fistula drainage, cholesterol 7alpha-hydroxylase (CYP7A1) mRNA and activity levels increased, FXR-mediated transcription was decreased, and FXR mRNA and nuclear protein levels declined. Replacing the enterohepatic bile acid pool with DCA restored FXR mRNA and nuclear protein levels and activated FXR-mediated transcription as evidenced by the increased expression of its target genes, SHP and BSEP, and decreased CYP7A1 mRNA level and activity. Replacing the bile acid pool with UCA also restored FXR mRNA and nuclear protein levels but did not activate FXR-mediated transcription, because the SHP mRNA level and CYP7A1 mRNA level and activity were unchanged. Feeding CA to intact rabbits expanded the bile acid pool enriched with the FXR high affinity ligand, DCA. FXR-mediated transcription became activated as shown by increased SHP and BSEP mRNA levels and decreased CYP7A1 mRNA level and activity but did not change FXR mRNA or nuclear protein levels. Thus, both hydrophobic and hydrophilic bile acids are effective in maintaining FXR mRNA and nuclear protein levels. However, the activating ligand (DCA) in the enterohepatic flux is necessary for FXR-mediated transcriptional regulation, which leads to down-regulation of CYP7A1.

Animals↗
PubMed2003

Inhibitory role of the somatostatin receptor SST2 on the intracrine-regulated cell proliferation induced by the 210-amino acid fibroblast growth factor-2 isoform: implication of JAK2.

The fibroblast growth factor (FGF)-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to grow in low serum concentrations through still unknown mechanisms called intracrine regulations. Different peptide hormones and cytokines have been found to be nuclearized through NLS and to induce cell proliferation. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. Pancreatic cells AR4-2J were stably transfected to express selectively the HMW FGF-2. We demonstrated that activation of the somatostatin receptor subtype SST2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates Galphai/JAK2/SHP-1. The Galphai inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-immunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with SST2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the following evidence: 1) the intracrine-induced proliferation can be reversed by extracellular acting polypeptides; 2) SST2 inhibitory signaling may involve the JAK2/SHP-1 pathway.

Animals↗
PubMed2003

Desensitization of signaling by oncostatin M in human vascular cells involves cytoplasmic Tyr residue 759 in gp130 but is not mediated by either Src homology 2 domain-containing tyrosine phosphatase 2 or suppressor of cytokine signaling 3.

Oncostatin M (OnM) signals through cell surface receptors, which utilize the gp130 subunit. In cultured human umbilical vein endothelial cells (HUVEC), OnM transiently elevates mRNA encoding for suppressor of cytokine signaling-3 (SOCS-3). By 1 h of OnM treatment, HUVEC become refractory to the restimulation by OnM, measured as failure to reinduce SOCS-3 mRNA. OnM-induced desensitization also prevents responses to other gp130-signaling cytokines (e.g. leukemia inhibitory factor and interleukin 11). OnM treatment does not affect gp130 expression levels and desensitizes signaling mediated by a transduced chimeric receptor containing extracellular domains of platelet-derived growth factor receptor-beta (PDGFRbeta) and the cytoplasmic region of gp130. Interestingly, a chimeric PDGFRbeta-gp130 mutant receptor, in which intracellular Tyr residue 759 of gp130 is replaced by a Phe residue, mediates prolonged signaling and is not cross-desensitized by OnM. Phospho-Tyr759 is the binding site for both SOCS-3 and for Src homology domain 2-containing tyrosine phosphatase 2 (SHP-2). In human aortic smooth muscle cells, neither prevention of SOCS-3 protein induction, using STAT3 or SOCS-3 antisense, nor prevention of SHP-2 expression, also with antisense, ablates desensitization. These data suggest that desensitization of vascular cells to OnM is mediated in trans and involves Tyr residue 759 in gp130 but is not mediated by either SOCS-3 or SHP-2, the only two proteins currently known to bind to gp130 at this site.

Animals↗
PubMed2003

Repression of farnesoid X receptor during the acute phase response.

The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 microg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response.

Acute-Phase Reaction↗