Biomedical subjects

M R Rosner

Publications and source records attributed to M R Rosner.

At least 55 records · Page 3Linked to original sources

PubMed1994

Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease.

We have shown that hyperoxic exposure of immature rats induces airway smooth muscle layer thickening and cell turnover parallel to that found in the airways of patients with bronchopulmonary dysplasia and chronic, severe asthma. We hypothesized that reactive oxygen species could promote the observed airway remodeling by directly stimulating signal transduction pathways that regulate cell growth. To test this hypothesis in cultured cells, we assessed the effects of hydrogen peroxide (H2O2) on mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes. The MAP kinases are a family of 40 to 46 kD cytosolic serine/threonine kinases that participate in the transduction of mitogenic signals to the cell nucleus. Quiescent cells were exposed to H2O2 (25 to 200 microns; 2 to 60 min), after which SDS-PAGE of cell extracts was performed. Western analysis using an anti-MAP kinase antiserum revealed a decrease in the mobility of the 42 and 44 kD MAP kinase bands after H2O2 exposures of 5 to 30 min, reflecting the phosphorylation at threonine and tyrosine residues required for enzymatic activity. MAP kinase activation was demonstrated by kinase renaturation assays, which showed an almost 4-fold increase in 42 and 44 kD MAP kinase activity. Down-regulation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) partially reduced H2O2-stimulated MAP kinase activity, suggesting that H2O2 induces MAP kinase activation via both PKC-dependent and PKC-independent pathways. Western analysis using a phosphotyrosine monoclonal antibody revealed increased tyrosine phosphorylation of proteins with approximate molecular weights of 72 and 125 kD after H2O2 exposure, demonstrating that H2O2 can stimulate the tyrosine phosphorylation of multiple cytosolic proteins, including MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Induction of epidermal growth factor receptor gene transcription by transforming growth factor beta 1: association with loss of protein binding to a negative regulatory element.

Transforming growth factor beta (TGF-beta) is a potent modulator of cell growth in many systems. In normal rat kidney fibroblasts, TGF-beta 1 increases epidermal growth factor (EGF) receptor gene transcription and synergizes with EGF to stimulate growth in soft agar, a characteristic of the transformed phenotype. In order to identify the target of TGF-beta 1 action, we have used a series of 5' deletion mutants of the EGF receptor promoter linked to a chloramphenicol acetyltransferase reporter gene (ERCAT). The TGF-beta response element(s) was localized to a cis-regulatory region which resides between positions -919 and -860 relative to the ATG translation initiation codon of the EGF receptor promoter. This 60-base pair region contains a repressor of the EGF receptor promoter and a TGF-beta inhibitory element that mediates TGF-beta 1 suppression of transin/stromelysin gene transcription through binding of a Fos-containing protein complex. Cotransfection of c-fos, c-jun, or both expression vectors with the intact or 5'-deleted ERCAT constructs identified several Fos-responsive inhibitory regions within the EGF receptor promoter, but these did not localize to the -919 to -860 promoter region. Mobility shift assays showed binding of the 60-base pair DNA fragment to proteins in extracts from untreated normal rat kidney cells; the binding was specifically competed by oligonucleotides containing a CAGATG sequence but not by oligonucleotides containing the EGF receptor repressor or the TGF-beta inhibitory element. TGF-beta 1 treatment but not anti-Fos antibody caused a decrease in specific 60-base pair DNA-protein complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Localization of immunoreactive epidermal growth factor receptor in neonatal and adult rat hippocampus.

The regional and developmental expression of epidermal growth factor (EGF) receptor in rat hippocampus was investigated utilizing immunocytochemical techniques at the light and electron microscopic levels. EGF receptor immunoreactivity in adult hippocampus was compared to that found at postnatal day 7 (P7). While the receptor was observed in P7 hippocampus, immunostaining was more prominent in the adult hippocampus, especially in the pyramidal CA2 field. Ultrastructural analysis of this region revealed that the receptor was localized to the cell bodies of both P7 and adult neurons rather than the axons or dendrites. The expression of EGF receptor in selected regions of the adult brain was verified by Western blotting. These results demonstrate the presence of EGF receptor in rat hippocampus as early as P7, localize the receptor to the pyramidal cell body, and establish the hippocampal formation, particularly CA2, as a major site of EGF receptor expression in rat brain.

Aging↗

PubMed1993

Functional analysis of conserved residues in the active site of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.

Amino Acid Sequence↗

PubMed1993

Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase.

Epidermal growth factor (EGF) receptor tyrosine kinase activity is down-regulated by a number of growth-modulating agents that activate protein kinase C and/or mitogen-activated protein (MAP) kinases. Although the mechanism is unclear, it has been hypothesized that phosphorylation of specific threonine residues leads to inhibition of the EGF receptor tyrosine kinase. Two sites phosphorylated on the EGF receptor in response to phorbol esters are possible mediators of this effect: threonine 654, the target of protein kinase C, and threonine 669, the target of MAP kinase and the major site of phosphorylation on the EGF receptor. In order to investigate the role of these residues in receptor regulation, we substituted glutamic acid to mimic the negative charge introduced by phosphorylation at these sites. The wild-type and mutant receptor cDNAs were then transfected into CHO cells that lack endogenous EGF receptor. The EGF binding properties of the mutant receptors were similar to those of the wild-type EGF receptors. EGF stimulated tyrosine kinase activity and DNA synthesis in cells expressing both mutant receptors, indicating that the mutant EGF receptors are biologically active. Treatment of cells with phorbol esters inhibited the high affinity EGF binding and tyrosine kinase activities of both mutant and wild-type EGF receptors. These results indicate that acidic residues at either the Thr-654 or Thr-669 site modulate but do not block EGF receptor signalling. Furthermore, this data demonstrates that the mutant EGF receptors are still a target for inhibition by phorbol esters. Thus, events other than phosphorylation of Thr-654 or Thr-669 appear to be required for receptor down-regulation by protein kinase C or MAP kinase.

Animals↗

PubMed1993

Mitogen-activated protein kinase regulates the epidermal growth factor receptor through activation of a tyrosine phosphatase.

Epidermal growth factor (EGF) receptor tyrosine kinase activity is inhibited by growth factor-stimulated kinases involved in cellular signaling. Mitogen-activated protein (MAP) kinase is activated in response to a wide variety of growth modulating agents including EGF. To determine whether MAP kinase can inactivate the EGF receptor tyrosine kinase, we investigated the effect of pp42 MAP kinase on the EGF receptor. The results indicate that direct phosphorylation of the EGF receptor by MAP kinase does not alter receptor tyrosine kinase activity. However, MAP kinase can decrease EGF receptor tyrosine phosphorylation through a vanadate-sensitive pathway involving activation of a tyrosine phosphatase.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1993

Mutations in a zinc-binding domain of human insulin-degrading enzyme eliminate catalytic activity but not insulin binding.

Insulin-degrading enzyme is a nonlysosomal metalloprotease that initiates degradation of internalized insulin in some cells. We previously identified a potential catalytic site containing an inversion of the Zn(2+)-binding domain of the thermolysin family (Kuo, W.-L., Gehm, B. D., and Rosner, M. R. (1991) Mol. Endocrinol. 4, 1580-1591). The role of this site in catalysis was examined by mutating one of the presumptive Zn(2+)-coordinating histidines (His108) in human insulin-degrading enzyme to leucine or glutamine, which were predicted to reduce or eliminate Zn2+ binding without substantially altering secondary structure. cDNAs for the mutant and wild-type enzymes were incorporated into an expression vector and transfected into COS cells. Expression of the transfected genes was confirmed by Northern and Western blots. In contrast to the wild-type gene, which increased insulin degradation by cell extracts and intact cells several-fold, the mutated genes had no effect on insulin degradation, indicating a loss of catalytic activity. However, the mutants' ability to bind substrate was unimpaired, as affinity labeling with 125I-insulin was increased compared to the wild type. These results suggest that an intact Zn(2+)-binding domain in human insulin-degrading enzyme is required for catalytic activity and can affect, but is not required for, substrate binding.

Amino Acid Sequence↗

PubMed1993

Multiple components of the T cell antigen receptor complex become tyrosine-phosphorylated upon activation.

Triggering of the multicomponent T cell antigen receptor (TCR) complex results in several biochemical processes which are critical for the functional activation of T lymphocytes. One common process is the tyrosine phosphorylation of several proteins, including the TCR zeta chain. Here we show that in addition to TCR zeta, other subunits (CD3 gamma, CD3 delta, and CD3 epsilon) of the TCR complex can also be tyrosine-phosphorylated in response to antigen receptor stimulation. This rapid phosphorylation was detected in several mature murine T cell subsets, including CD4+ type 1 and 2 helper cells (TH1 and TH2). Therefore, tyrosine phosphorylation of multiple TCR components in addition to TCR zeta may be an important event during the initiation of the signaling cascade leading to T cell activation.

Animals↗

PubMed1993

Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking.

The ligand-activated tyrosine kinase receptor for epidermal growth factor (EGF) is down-regulated by an integral membrane protein coded for by the E3 early transcription unit of group C adenoviruses. The E3 protein appears to block recycling of constitutively internalized receptors, causing them instead to traffic to lysosomes where they are degraded. Expression of functional EGF receptors is also regulated by protein kinase C (PKC), which directly phosphorylates the EGF receptor at Thr-654. The goal of this study was to determine potential interactions between PKC and the E3 protein, since membrane-bound PKC activity is elevated by the adenovirus E1A protein. Our results show that although tumor promoters which activate PKC cause a coordinate induction of E3 protein synthesis and EGF receptor degradation, the E3 protein-induced pathway for receptor down-regulation functions independently of PKC and other kinases that are inhibited by staurosporine. This suggests that in contrast to other mechanisms that modulate receptor expression (i.e., ligand and PKC), the E3 protein is not regulated by phosphorylation but is constitutively active. We also report that adenovirus-mediated degradation is the preferred pathway in infected cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce receptor recycling.

3T3 Cells↗

PubMed1993

Activation of mitogen-activated protein kinase by epidermal growth factor in hippocampal neurons and neuronal cell lines.

Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.

Animals↗

PubMed1993

Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues.

Insulin-degrading enzyme (IDE), a cytosolic metalloendoprotease, can degrade insulin, insulin-like growth factor-II, insulin-like growth factor-I, and transforming growth factor-alpha. While IDE has been implicated in the cellular degradation of insulin, other physiological functions of this enzyme are not known. To assess the possible role of IDE in cellular growth and development, we determined the tissue and developmental distribution of the enzyme. Rat IDE cDNA fragments and antibodies directed against human IDE were used to probe IDE transcripts and proteins in rat tissues. The results demonstrate that IDE transcripts are ubiquitous in rat tissues. The level of rIDE transcripts is high in adult rat testis, tongue, and brain; moderate in kidney, prostate, heart, muscle, liver, intestine, and skin; and low in spleen, lung, thymus, and uterus. The sizes of the major transcripts of rIDE are 3.4 and 6.3 kilobases in all tissues analyzed, except testis. Surprisingly, the highest level of rIDE mRNA in the adult rat was in the testis, and the major transcripts of rIDE in this tissue were shifted in size to 3.8 and 6.7 kilobases. Immunocytochemical analysis localized the rIDE mainly in the epithelium of prostate gland and kidney, and the cytosol of liver hepatocytes. During rat development from 6-7 days of age to adulthood, rIDE mRNA levels increased in brain, testis, and tongue; decreased in muscle and skin; and did not significantly change in other tissues examined. These studies reveal regulation of IDE or IDE-related genes in rat tissues and during rat development, suggesting that this enzyme may have multiple functions relating to cellular growth and development.

Aging↗

PubMed1992

Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor.

In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.

Blotting, Western↗

PubMed1992

Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression.

Clonal cell lines of rat embryonic hippocampal origin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brain-derived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.

3T3 Cells↗

PubMed1992

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity.

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.

Animals↗

PubMed1991

Epidermal growth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells is an EGF-stimulated "MAP" kinase.

The epidermal growth factor (EGF) receptor is both an activator and a target of growth factor-stimulated kinases involved in cellular signaling. Threonine-669 (T669) of the EGF receptor is phosphorylated in response to a wide variety of growth-modulating agents. MAP kinase is similarly phosphorylated as well as stimulated by growth activators, including EGF. To determine whether a MAP-type kinase is responsible for T669 kinase activity in EGF-stimulated 3T3-L1 cells, we partially purified and characterized the T669 peptide kinase. The results indicate that a MAP kinase phosphorylates the T669 peptide and raise the possibility that this enzyme may participate in a feedback loop, being activated by the EGF receptor and in turn phosphorylating the receptor.

Animals↗

PubMed1991

Localization of the gene encoding insulin-degrading enzyme to human chromosome 10, bands q23----q25.

Insulin-degrading enzyme (IDE) is a cytosolic proteinase involved in the cellular processing of insulin. Using somatic cell hybrid analysis and in situ chromosomal hybridization, we have localized the gene encoding IDE to human chromosome 10, bands q23----q25. The murine Ide gene was previously mapped to Chromosome 19; together, these results suggest that the IDE gene is a member of a conserved syntenic group on human chromosome 10, bands q23----q25 and mouse Chromosome 19.

Animals↗

PubMed1991

Regulation of insulin, epidermal growth factor, and transforming growth factor-alpha levels by growth factor-degrading enzymes.

The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.

Bacitracin↗

PubMed1991

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.

Ammonium Chloride↗