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L M Wang

Publications and source records attributed to L M Wang.

At least 73 records · Page 4Linked to original sources
PubMed1996

IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway.

Although it has been known for some time that IL-4 protects cells from death via apoptosis, very little is known about the mechanism by which IL-4 exerts this effect. In this report, we have studied the molecular mechanisms of the IL-4-induced prevention of apoptosis in the 32D and B cell systems. IL-3 withdrawal has been shown to induce G1-arrest in 32D cells and subsequent death by apoptosis. We found that overexpression of IRS-1 protected 32D cells from death induced by IL-3 deprivation. IL-4 was able to protect 32D cells from apoptosis in the presence or absence of IRS-1. However, the protection from apoptosis of cells cultured in IL-4 was greater in the presence of IRS-1 and it reached levels comparable to those of cells cultured in IL-3. The IRS-1-dependent prevention of apoptosis is linked to the activation of PI 3'-kinase since wortmannin and LY294002, two inhibitors of PI 3'-K, partially inhibited the prevention of apoptosis mediated by IL-4 in 32D-IRS-1 cells after IL-3 withdrawal but not in 32D cells lacking IRS-1 expression. In addition, we found that the IRS/PI 3'-K pathway is, at least in part, responsible for the prevention of apoptosis by IL-4 in normal splenic B cell cultures. In spite of the ability of murine IL-4 to partially protect 32D cells lacking IRS-1 from apoptosis, human IL-4 was not able to prevent cell death in 32D-IRS-1 cells transfected with the human IL-4 receptor mutated in the insulin-lL-4 receptor motif (14R-motif) at amino acid 497 (Y497F). This mutation has previously been shown to abrogate the tyrosine phosphorylation of IRS-1 by human IL-4. These results demonstrate that IL-4 protects 32D cells from apoptosis by two different pathways, one of them mediated by IRS-1. In addition, these results suggest that the 14R-motif of the IL-4R is linked to both pathways.

Animals↗
PubMed1996

The tau mutation shortens the period of rhythmic photoreceptor outer segment disk shedding in the hamster.

The outer segments of vertebrate retinal photoreceptors undergo periodic shedding of membrane from their distal tips. This circadian rhythm of disk shedding persists with a period of about 24 h in the absence of external time cues. A circadian oscillator controlling photoreceptor disk shedding may exist in the eye, but in addition, the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) may also influence ocular rhythms including that of disk shedding. The tau mutation directly affects the SCN, and shortens the period of locomotor activity from 24 h in wild-type hamsters to 20 h in homozygous mutants. Here we show that homozygous tau-mutant hamsters in a 20-h light/dark cycle exhibit a 20-h oscillation in the rate of disk shedding, with peak phagosome numbers in the retinal pigmented epithelium occurring just after light onset. The numbers of phagosomes are significantly elevated from mid-dark levels prior to light onset, indicating that the disk shedding cycle anticipates dawn. Under conditions of constant darkness, the disk shedding rhythm in tau-mutant hamsters persists with a period of approximately 20 h. These results indicate that a rhythm of retinal photoreceptor outer segment disk shedding exists in the hamster eye, and that the period of this rhythm is shortened by the tau mutation.

Adaptation, Ocular↗
PubMed1996

Stat6 and Jak1 are common elements in platelet-derived growth factor and interleukin-4 signal transduction pathways in NIH 3T3 fibroblasts.

Both platelet-derived growth factor (PDGF) and interleukin-4 (IL-4) play major roles in cell proliferation, differentiation, chemotaxis, and other functional responses. Here, we demonstrate that Stat6, previously shown to be activated by only IL-4 and IL-3, becomes activated after PDGF stimulation of NIH 3T3 fibroblasts. PDGF BB, and to a lesser extent PDGF AA, rapidly induced DNA binding activity from NIH 3T3 cell lysates utilizing the immunoglobulin heavy chain germ line epsilon promoter (Iepsilon) that specifically binds to Stat6 in an electrophoretic mobility shift assay. DNA binding activity could be detected within 5 min and reached maximum levels at approximately 20 min in parental NIH 3T3 cells. An identical mobility shift and time course of PDGF-mediated Iepsilon binding activity was more pronounced in lysates of NIH 3T3 transfectants overexpressing human Stat6 (NIH 3T3-Stat6). The observed radiolabeled Iepsilon mobility shift was competed by unlabeled Iepsilon as well as by the beta-casein gene promoter but not by the interferon-alpha-stimulated response element or the interferon-gamma response region of the guanylate-binding protein gene. A Stat6-specific polyclonal antisera also supershifted the PDGF-induced Iepsilon mobility shift. After PDGF BB treatment, a 100-kDa tyrosine phosphorylated species was detected in anti-Stat6 immunoprecipitates. Cycloheximide had little effect on Stat6 tyrosine phosphorylation. In addition to Stat6, Stat5a, and Stat5b, PDGF BB also induced Jak1 tyrosine phosphorylation suggesting a potential pathway for Stat activation. Strikingly, the concurrent addition of IL-4 enhanced PDGF BB-induced Iepsilon binding activity, Jak1 tyrosine phosphorylation, and [3H]thymidine incorporation. These results provide evidence that Stat6 and Jak1 are common elements in PDGF and IL-4 signaling pathways and suggest that IL-4 could play a role in potentiating certain known PDGF-induced biological responses.

3T3 Cells↗
PubMed1996

Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors. Evidence for two distinct phosphotyrosine-dependent interaction domains within IRS-2.

Insulin receptor substrate 2 (IRS-2) has recently been shown to be a substrate of the insulin receptor (IR). In this study we utilize the yeast two-hybrid system and assays of in vitro interaction to demonstrate that IRS-2 interacts directly with the IR and the insulin-like growth factor I receptor. We show that, like IRS-1, the region of IRS-2 that contains the putative phosphotyrosine binding and SAIN elements (188-591) is sufficient for receptor interaction and that this interaction is dependent upon the NPX(p)Y (where (p)Y is phosphotyrosine) motifs within the juxtamembrane domains of the receptors. In addition to this amino-terminal NPX(p)Y-binding domain, an additional domain of strong interaction was identified in the central region of IRS-2 and was localized between amino acids 591 and 733. This interaction was found to be dependent upon receptor phosphorylation but was NPX(p)Y-independent. This region does not appear to have either an SH2 or a phosphotyrosine binding domain. Both of the interactions could also be demonstrated in vitro using IRS-2 glutathione S-transferase fusion proteins. We conclude that IRS-2, unlike IRS-1, can interact with tyrosine-phosphorylated receptors such as the IR and insulin-like growth factor I receptor via multiple independent binding motifs. Our findings suggest the existence of a previously unidentified phosphotyrosine-dependent binding domain within the central region of IRS-2.

Animals↗
PubMed1996

The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

3T3 Cells↗
PubMed1996

Preventive effects of unsei-in and oren-gedoku-to, Chinese traditional medicines, against rat paw oedema and abdominal constriction in mice.

Oren-gedoku-to and Unsei-in are complex mixtures of ingredients derived from plants. These two drugs are clinically most frequently used in the treatment of Behçet's disease, we have investigated the anti-inflammatory and analgesic actions of Oren-gedoku-to and Unsei-in using a battery of tests; rat paw oedema induced by five different agents; abdominal constriction; mouse ear swelling; and dye leakage tests, designed to clarify the therapeutic potential of these medicines. The findings in this study that these medicines are able to inhibit the rat paw oedema, suppress the abdominal constriction and inhibit the increased dye permeability confirm the analgesic and anti-inflammatory effects of these compounds. From these results there seems a clear rationale for exploring the effectiveness of these Chinese medicines in the treatment of chronic and acute inflammatory diseases.

Abdomen↗
PubMed1996

YMXM motifs and signaling by an insulin receptor substrate 1 molecule without tyrosine phosphorylation sites.

Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) by the activated receptors for insulin, IGF-1, and various cytokines creates binding sites for signaling proteins with Src homology 2 domains (SH2 proteins). Determining the role of specific SH2 proteins during insulin signaling has been difficult because IRS-1 possesses as many as 18 potential tyrosine phosphorylation sites, several of which contain redundant motifs. Using 32D cells, which contain no endogenous IRS proteins, we compared the signaling ability of an IRS-1 molecule in which 18 potential tyrosine phosphorylation sites were replaced by phenylalanine (IRS-1(F18)) with two derivative molecules which retained three YMXM motifs (IRS-1(3YMXM)) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1(YCT)). During insulin stimulation, IRS-1(F18) failed to undergo tyrosine phosphorylation or mediate activation of the phosphotidylinositol (PI) 3'-kinase or p70(s6k); IRS-1(YCT) was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1(3YMXM) nor IRS-1(YCT) mediated activation of mitogen-activated protein kinases. IRS-1(F18) and IRS-1(YCT) partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-l(YCT) do not augment this signal. IRS-1(3YMXM) mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1(3YMXM) with PI 3'-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3'-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.

Amino Acid Sequence↗
PubMed1996

Seasonal variation in the incidence of peptic ulcer and esophageal variceal bleeding in Taiwan.

BACKGROUND: Seasonal variation of peptic ulcer and peptic ulcer bleeding has been reported in many western countries. To investigate the seasonal variation of peptic ulcer (PU) and esophageal variceal (EV) bleeding in Taiwan, this retrospective study was conducted. METHODS: Seven hundred and forty-six cases of gastric ulcer (GU) bleeding, 777 cases of duodenal ulcer (DU) bleeding and 264 cases of EV bleeding were recruited from January 1, 1991 to December 31, 1992. Patients were sent to the Medical Emergency Room (MER) for hematemesis and/or melena. Upper gastrointestinal (UGI) endoscopic examinations were completed within 24 hours for all patients. RESULTS: Significant seasonal variation was found in the incidence of PU and EV bleeding. PU bleeding was most often seen in March and EV bleeding was most often seen in February. CONCLUSIONS: The possible mechanism for the cyclic change of PU and EV bleeding is unclear, but clearly more manpower is needed during the months of February and March for better management of the increasing number of cases of PU and EV bleeding.

Aged↗
PubMed1996

[An animal model for screening of antiallergic and antipruritic drugs].

4-Aminopyridine(4-AP) 1 mg.kg-1 sc at the scruff induced a licking response in mice. Antiallergic and antipruritic drugs, such as diphenhydramine HCl(20 mg.kg-1 ip), doxepin(12.5 mg.kg-1 ig), prednisone(10 mg.kg-1 ig), dexamethasone(10 mg.kg-1 ip), fluocinolone (applied to the surface of skin), Pi Yan Ping(applied to the surface of skin), disodium cromoglicate(400 mg.kg-1 ip), ketotifen(1 mg.kg-1 ip), etc. markedly inhibited the licking response elicited by 4-AP. The calcium antagonist nifedipine(500 mg.kg-1 lg) and the potassium channel opener minoxidil(400 mg.kg-1 ig) produced the same inhibitory effect. H2-receptor antagonist cimetidine(200 mg.kg-1 ip) and ranitidine(150 mg.kg-1 ip) showed no effect. Morphine HCl(10 mg.kg-1 ip) and diazepam(0.02 mg.kg-1 ip) exhibited antagonistic effect on the licking response induced by 4-AP, but phenobarbital(25 mg.kg-1 ip), pentobarbital(15 mg.kg-1 ip) and aspirin(300 mg.kg-1 ig) did not. These results indicate that many antiallergic or antipruritic drugs inhibited the licking response induced by 4-AP. The method of licking response elicited by 4-AP has the merit of simplicity and convenience and may be used for screening antiallergic and antipruritic drugs.

4-Aminopyridine↗
PubMed1996

[Effect of norepinephrinergic system on ipalbidine analgesia].

Ipalbidine (Ipa) is a photoactive alkaloid isolated from the seeds of ipomoea hardwickki Hemsl. The analgesic effects of Ipa were determined by rat tail flick method. A dose-dependent analgesic effect was found after s.c. or i.c.v. administration of Ipa, but no analgesia was observed after intrathecal injection, indicating that the analgesic effect of Ipa is central in origin, and it acts mainly on supraspinal substrate. The analgesic effect induced by Ipa (60 mg.kg-1, s.c.) was markedly reduced by reserpine (2 mg.kg-1, i.p.) given 24 h before Ipa, which was reversed by combined administration with i.c.v. norepinephrine (NE). In addition, Ipa-induced analgesia was significantly attenuated by electrolytic lesion of bilateral destruction of locus coeruleus, and combined administration with diethyldithiocarbamate (200 mg.kg-1, i.p.), phentolamine (10 mg.kg-1, i.p. or i.c.v.), and prazosin (3 mg.kg-1, s.c.). But no influence was observed on the analgesia of Ipa after administration of yohimbine (5 mg.kg-1, s.c.) or propranolol (10 mg.kg-1, i.p.). These results suggest that the analgesia caused by Ipa is closely related to the function of the central norepinephrinergic system, and probably mediated by indirectly acting on alpha 1 receptors, but not alpha 2 or beta receptors.

Analgesics, Non-Narcotic↗
PubMed1995

Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

Humans↗
PubMed1995

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Role of IRS-2 in insulin and cytokine signalling.

The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

Amino Acid Sequence↗
PubMed1995

An unexpected transforming gene in calf-thymus carrier DNA: bovine hst.

During a search for transforming genes by transfecting a human cDNA expression library together with calf thymus carrier-DNA into NIH/3T3 cells, we found a focus which was induced by a plasmid containing a sequence highly homologous to human HST (a transforming gene from Human STomach cancer). However, PCR analysis identified the source of this sequence as calf thymus DNA. The deduced amino acid (aa) sequence of bovine HST shows 91 and 81% identity to the human and mouse HST aa sequences, respectively. These data suggest that the hst of calf thymus carrier-DNA could induce transformation of NIH/3T3 cells.

3T3 Cells↗
PubMed1995

Structural studies on the PH domains of Db1, Sos1, IRS-1, and beta ARK1 and their differential binding to G beta gamma subunits.

Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The pleckstrin homology domain in insulin receptor substrate-1 sensitizes insulin signaling.

The NH2 terminus of insulin receptor substrate-1 (IRS-1) contains a pleckstrin homology (PH) domain. We deleted the PH domain in IRS-1 (IRS-1 delta PH) and expressed the mutant in Chinese hamster ovary and 32D cells. During insulin stimulation, IRS-1 delta PH is poorly tyrosine-phosphorylated in CHO cells, but undergoes serine/threonine phosphorylation. Similarly, IRS-1 delta PH fails to undergo insulin-stimulated tyrosine phosphorylation in 32D cells, which uncouples the activation of phosphatidylinositol 3'-kinase and p70s6k from the endogenous insulin receptors. Overexpression of the insulin receptor in 32DIR cells, however, restores tyrosine phosphorylation of IRS-1 delta PH and rescues insulin responses including mitogenesis. Thus, while the PH domain is not required for the engagement of downstream signals, it is one of the elements in the NH2 terminus of IRS-1 that is needed for a sensitive coupling to insulin receptors, especially at ordinary receptor levels found in most cells and tissues.

Animals↗
PubMed1995

Differential requirement of a motif within the carboxyl-terminal domain of alpha-platelet-derived growth factor (alpha PDGF) receptor for PDGF focus forming activity chemotaxis, or growth.

To determine the molecular basis for the transforming function of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have constructed chimerae consisting of the extracellular domain of the human CSF-1R (fms) linked to the cytoplasmic domain of the alpha PDGF receptor (alpha R) containing a series of deletion or point mutations. The ability of fms/alpha R chimerae to mediate CSF-1-dependent anchorage-independent growth, focus formation, and chemotaxis of NIH/3T3 cells was then examined. Our results provide evidence that a domain encompassing amino acid residues 977-1024 of the alpha PDGFR is required for ligand-dependent focus formation, but not chemotaxis or anchorage-independent growth, and that tyrosine residues within this domain constitute the major binding site for phospholipase C gamma. Therefore, our findings suggest that: (i) the focus forming function of alpha PDGFR correlates well with the ability of the receptor to bind phospholipase C gamma, and (ii) the mechanism of focus formation mediated by alpha PDGFR may be distinguished from that required for chemotaxis or anchorage-independent growth.

3T3 Cells↗