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Biomedical subjects

S Dedhar

Publications and source records attributed to S Dedhar.

At least 55 records · Page 3Linked to original sources
PubMed1997

Protein kinase mediators of integrin signal transduction.

Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of 'solid-state' signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion.

Animals↗
PubMed1996

New mechanisms of regulation of the genomic actions of vitamin D in bone cells: interaction of the vitamin D receptor with non-classical response elements and with the multifunctional protein, calreticulin.

Vitamin D exerts its genomic effects following binding to a specific receptor which is a member of the steroid hormone receptor superfamily. The vitamin D receptor (VDR) forms heterodimers with retinoid X receptors (RXRs) and the dimer then interacts with its cognate binding site, termed vitamin D response element (VDRE), to affect the transcription of target genes. Recent studies have identified novel sequence motifs for VDREs as well as novel protein-protein interactions involving the VDR. These will be reviewed with particular emphasis on the complex VDRE from the c-fos proto-oncogene promoter region and the inhibition of the vitamin D signal transduction pathway by the multifunctional protein, calreticulin. Thus research on the control of gene transcription by vitamin D reveals examples of molecular interplay between transcriptional regulatory pathways and provides new insight into the molecular mechanism of action of vitamin D.

Base Sequence↗
PubMed1996

Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.

The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. We used a two-hybrid screen to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encoded a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. We report here that this integrin-linked kinase (ILK) phosphorylated a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitated with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity was reduced in response to fibronectin. Overexpression of p59ILK disrupted epithelial cell architecture and inhibited adhesion to integrin substrates, while inducing anchorage-independent growth. We propose that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction.

Amino Acid Sequence↗
PubMed1996

Integrin cytoplasmic interactions and bidirectional transmembrane signalling.

Integrins are heterodimeric integral plasma membrane proteins containing extracellular, transmembrane, and cytoplasmic domains. These highly versatile receptors mediate not only cell adhesion and migration, but also the bidirectional transfer of information across the plasma membrane. The cytoplasmic domains of integrins are required for the transduction of this bidirectional information, and have recently been shown to participate in direct interactions with some novel cytoplasmic proteins, such as an ankyrin repeat containing serine/threonine protein kinase (integrin-linked kinase) and beta3 endonexin. New evidence also suggests that, via interactions with focal adhesion kinase, the integrin cytoplasmic domains can coordinate actin cytoskeletal organization and responses to growth factors. The elucidation of the signal transduction pathways activated by integrins is an intense area of investigation that has shown that integrins have some unique properties as signal transducing receptors.

Amino Acid Sequence↗
PubMed1996

Association of 1 alpha,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites.

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], 25-hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1 alpha,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone-occupied VDR. These results were substantiated by studies using baculovirus-expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1 alpha,25-(OH)2D3.

Animals↗
PubMed1995

Inducible interaction of integrin alpha 2 beta 1 with calreticulin. Dependence on the activation state of the integrin.

We have previously demonstrated an interaction between the highly conserved KXGFFKR sequence of the integrin alpha-subunit cytoplasmic domains and calreticulin. Since this highly conserved sequence motif has been implicated in the regulation of the integrin affinity state, we wanted to determine whether the calreticulin-integrin interaction also depended on the integrin affinity state, and whether calreticulin occupation of integrin via the KXGFFKR motif was involved in the regulation of the ligand affinity state. We now demonstrate that anti-integrin antibody- or phorbol 12-myristate 13-acetate (PMA)-induced activation of the alpha 2 beta 1 integrin on Jurkat cells, as determined by stimulation of adhesion to collagen type I, resulted in an increased amount of calreticulin bound to this integrin. alpha 2 beta 1 activation with either anti-beta 1 or anti-alpha 2 monoclonal antibodies resulted in a greater amount of calreticulin coimmunoprecipitating with this integrin. Inactivation by neutralizing anti-integrin antibodies abrogated the calreticulin-integrin interaction. A correlation was also found between PMA-induced alpha 2 beta 1 activation and the amount of calreticulin bound to this integrin. Furthermore, pretreatment of streptolysin O-permeablized Jurkat cells with an anti-calreticulin antibody resulted in a significant and specific inhibition of the adhesion to collagen type I that could be induced by antibodies to alpha 2 beta 1 or by PMA. These data suggest that the active, high affinity form of alpha 2 beta 1 binds calreticulin and that calreticulin binding to the alpha 2 cytoplasmic domain may be required for stabilizing the high affinity state of this integrin. The data presented here also demonstrate, for the first time, an inducible interaction of an integrin with an intracellular protein that occurs via the alpha subunit of the integrin.

Amino Acid Sequence↗
PubMed1995

Identification of a novel form of the alpha 3 integrin subunit: covalent association with transferrin receptor.

The alpha 3 beta 1 integrin is a cell-surface receptor for laminin, entactin, collagen, fibronectin and epiligrin. On some prostatic-carcinoma cell lines that express the alpha 3 beta 1 heterodimer we have identified a novel form of the alpha 3 subunit. Whereas the prototypic alpha 3 subunit has a molecular mass of approximately 155 kDa, we have isolated a approximately 225 kDa protein (p225) which is recognized by monoclonal antibodies to the alpha 3 subunit. Protein sequence analysis revealed that p225 consists of two polypeptides, namely integrin alpha 3 heavy chain (approximately 130 kDa) disulphide-bonded to a monomer of the transferrin receptor (approximately 95 kDa) instead of the typical alpha 3 light chain (approximately 25 kDa). The p225 seems to be directly associated with beta 1 subunit, since it was immunoprecipitable with anti-(beta 1 subunit) antibodies. The association of transferrin receptor and integrin alpha 3 was apparently not the result of spurious disulphide-bond formation occurring during the protein purification, as iodoacetamide and GSH did not block the formation of the complex. The transferrin receptor is normally a homodimer that is involved in the internalization of iron-bound transferrin into cells and can be expressed at relatively high levels in the cell lines which we have studied. The p225 is not found on all cell types examined to date and therefore it may represent a unique complex between the integrin alpha 3 subunit and the transferrin receptor, a covalent association which may play a role in the adherence and/or proliferation of some types of tumour cells.

Amino Acid Sequence↗
PubMed1995

Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V.

Malignant transformation of fibroblast and epithelial cells is accompanied by increased beta 1-6 N-acetylglucosaminyltransferase V (GlcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GlcNAc-TV in Mv1Lu cells, an immortalized lung epithelial cell line results in loss of contact-inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme alpha-mannosidase II. In serum-deprived and high density monolayer cultures, the GlcNAc-TV transfectants formed foci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GlcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GlcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface alpha 5 beta 1 or alpha v beta 3 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins alpha 5, alpha v and beta 1 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that beta 1-6GlcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells.

Actin Cytoskeleton↗
PubMed1995

Constitutive expression of calreticulin in osteoblasts inhibits mineralization.

Recent studies have shown that the multifunctional protein calreticulin can localize to the cell nucleus and regulate gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors. A number of known modulators of bone cell function, including vitamin D, act through this receptor family, suggesting that calreticulin may regulate their action in bone cells. We have used a gain-of-function strategy to examine this putative role of calreticulin in MC3T3-E1 osteoblastic cells. Purified calreticulin inhibited the binding of the vitamin D receptor to characterized vitamin D response elements in gel retardation assays. This inhibition was due to direct protein-protein interactions between the vitamin D receptor and calreticulin. Expression of calreticulin transcripts declined during MC3T3-E1 osteoblastic differentiation. MC3T3-E1 cells were transfected with calreticulin expression vectors; stably transfected cell lines overexpressing recombinant calreticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Constitutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expression was unaffected. This pattern mimicked the gene expression pattern observed in parental cells before down-regulation of endogenous calreticulin expression. In long-term cultures of parental or vector-transfected cells, 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3) induced a two- to threefold stimulation of 45Ca accumulation into the matrix layer. Constitutive expression of calreticulin inhibited the 1,25(OH)2D3-induced 45Ca accumulation. This result correlated with the complete absence of mineralization nodules in long-term cultures of calreticulin-transfected cells. These data suggest that calreticulin can regulate bone cell function by interacting with specific nuclear hormone receptor-mediated pathways.

Amino Acid Sequence↗
PubMed1995

Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of alpha 3 and alpha 6 integrins.

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of alpha 3 beta 1 and alpha 6 beta 1/alpha 6 beta 4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the alpha 3 beta 1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-alpha 3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the alpha 6 beta 1 integrin on these cells with anti-alpha 6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of alpha 5 beta 1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-beta 1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-beta 4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both alpha 6 beta 1 and alpha 6 beta 4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-alpha 3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin heterodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.

Antibodies↗
PubMed1994

Inhibition of nuclear hormone receptor activity by calreticulin.

We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.

Amino Acid Sequence↗
PubMed1994

The effect of GM-CSF and G-CSF on the growth of human osteosarcoma cells in vitro and in vivo.

The human osteosarcoma cell line, MG63, responds both to GM-CSF and to G-CSF in vitro. To assess the significance of these observations to tumor growth in vivo, MG63 cells were engineered by retroviral infection to produce human GM-CSF or G-CSF. These retrovirally infected cells become autostimulatory as measured by increased [3H]-thymidine incorporation (3- to 7-fold) and anchorage-independent colony formation (7- to 10-fold) as compared with uninfected MG63 cells or cells infected with control (neor) retrovirus. The increased proliferation induced by exogenous GM-CSF or G-CSF on uninfected MG63 cells in both assays could be completely inhibited by anti-GM-CSF or anti-G-CSF antibodies, while the same antibodies only partially abrogated proliferation by the growth-factor-producing cells. None of 34 nude or SCID mice developed tumors when injected s.c. with uninfected or neor-virus-infected cells. In contrast, all 30 mice injected with GM-CSF- or G-CSF-producing MG63 cells developed tumors which were G418-resistant and factor-producing. Tumor cell DNA showed a polyclonal retroviral integration pattern indistinguishable from that in the DNA of cells injected into mice. Tumors that formed following injection of a mixture of G418-resistant, GM-CSF-producing cells and cells infected with virus containing only the hygror gene contained hygromycin-resistant cells in the same proportion as was present in the original cell mixture. These data indicate that GM-CSF and G-CSF can support the growth of an osteosarcoma cell line both in vitro and in vivo whether the factor is supplied by autocrine production or from exogenous sources.

3T3 Cells↗
PubMed1994

Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix.

The human ovarian surface epithelium (OSE), or ovarian mesothelium, is functionally complex as seen by its capacity to proliferate, migrate, and contribute to ovulation and ovulatory repair in response to cyclic hormonal and environmental changes. We wished to determine whether this phenotypic versatility is reflected in cell-extracellular matrix interactions in primary and low-passage culture. Comparisons of cultures maintained on different substrata revealed that these cells form cohesive monolayers on plastic, while fibrin clots enhance cell dispersion, and thus may provide a migratory cue. The cells invaded Matrigel as multicellular aggregates, while collagen gels mediated a morphologic epithelial-mesenchymal conversion. On plastic, the cells produced extracellular matrix components characteristic of epithelial basement membrane (laminin and collagen type IV), as well as stroma (collagen types I and III). In addition, ovarian surface epithelial cells secreted serine proteases and matrix metalloproteinases. The levels of chymotrypsin- and elastase-like proteases were dictated by the substratum: low levels were secreted by cells grown on plastic, intermediate levels on collagen gels and fibrin clots, and most protease was produced on Matrigel. The rate of cell proliferation varied with the substrata and was inversely related to protease secretion. Integrin expression was greatest on plastic and least on collagen gels where integrins were downregulated with time. alpha 6/beta 4 was absent from all cells while varying levels of alpha 2, alpha 3, alpha 5, beta 1, and vitronectin receptor were detected depending on the culture substratum employed. In low-passage cultures of human ovarian surface epithelial cells, then, cell shape, growth, protease production, and integrin expression are modulated by the extracellular matrix. The cells, in turn, alter extracellular matrix by synthesis, lysis, and physical remodeling, and express both stromal and epithelial characteristics. The broad repertoire of these functions may be related to their mesodermal origin, and may reflect the expression of a dual, epithelio-mesenchymal phenotype by relatively immature, uncommitted cells. The results demonstrate the great complexity and versatility of these interactions which render OSE cells capable of participating in numerous physiological and pathological processes.

Adult↗
PubMed1994

Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin alpha-subunit-binding protein.

We have demonstrated recently that calreticulin, an intracellular calcium-binding protein, can interact with the alpha-subunits of integrin receptors via the highly conserved KXGFFKR amino acid sequence present in the cytoplasmic domains of all integrin alpha-subunits (Rojiani et al. (1991) Biochemistry 30, 9859-9866). Here we demonstrate that calreticulin can be co-localized by immunofluorescence as well as co-purified with integrins, that recombinant calreticulin can also interact with integrins, and that the interaction occurs predominantly via the N-domain of calreticulin, to a much lesser extent with the C-domain, but not at all with the proline-rich P-domain. To demonstrate a physiological role for the interaction of calreticulin with integrins, calreticulin expression was downregulated by treating cells with antisense oligonucleotides designed to inhibit the initiation of translation of calreticulin. Antisense oligonucleotides, but not sense or non-sense oligonucleotides, inhibited attachment and spreading of cells cultured in the presence of fetal bovine serum, and also of cells plated on individual extracellular matrix substrates in the absence of serum. The antisense oligonucleotide inhibited cell proliferation of anchorage-dependent cells slightly, but there was no effect on cell viability. The effect on cell attachment was similar to that achieved by treating cells with an antisense oligonucleotide designed to inhibit translation of the integrin alpha 3 subunit, which resulted in the inhibition of cell attachment to alpha 3 beta 1-specific substrates. The effect of the antisense calreticulin oligonucleotide on cell attachment was demonstrated to be integrin-mediated since antisense calreticulin treatment of Jurkat cells abrogated the stimulation of collagen cell attachment achieved by attachment-stimulating signalling anti-alpha 2 (JBS2) and anti-beta 1 (21C8) antibodies. The oligonucleotides did not affect the rate of cell proliferation of these cells. These results demonstrate a fundamental role of calreticulin in cell-extracellular matrix interactions.

Amino Acid Sequence↗
PubMed1993

Stimulation of tyrosine phosphorylation and accumulation of GTP-bound p21ras upon antibody-mediated alpha 2 beta 1 integrin activation in T-lymphoblastic cells.

Integrins are a family of heterodimeric integral plasma membrane proteins that behave as receptors for components of the extracellular matrix and also mediate cell to cell adhesion. Occupation of integrins can result in the transduction of intracellular signals, leading to cytoskeletal reorganization, tyrosine phosphorylation, and induction of gene expression. We report here that the ligation of alpha 2 beta 1 integrin by collagen-adhesion stimulatory anti-alpha 2 and anti-beta 1 antibodies resulted in the accumulation of p21ras in the active GTP-bound state in Jurkat T-lymphoblastoid cells. The activation was accompanied by the tyrosine phosphorylation of proteins of 47-52 kDa. This stimulation of tyrosine phosphorylation and p21ras activation was specific for the activating antibodies and occurred within 2 min of the addition of these antibodies. Although treatment of the cells with the protein kinase C activator, phorbol 12-myristate 13-acetate also resulted in an induction of both cell attachment to collagen and of p21ras activation, tyrosine phosphorylation was not observed. These results demonstrate that alpha 2 beta 1 integrin activation can result in the specific stimulation of tyrosine phosphorylation of 47-52-kDa proteins, as well as activation of a signaling pathway involving p21ras.

Antibodies↗
PubMed1993

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗