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Biomedical subjects

T O Daniel

Publications and source records attributed to T O Daniel.

At least 37 records · Page 2Linked to original sources
PubMed1993

Spatial organization of the extracellular matrix modulates the expression of PDGF-receptor subunits in mesangial cells.

The aim of this study was to test the hypothesis that changes in the extracellular matrix environment regulate rat mesangial cell growth by modulation of the expression of both PDGF-receptor alpha- and beta-subunits. We investigated the mitogenic effects of the PDGF isoforms AA, AB and BB in conventional two-dimensional (2D) culture on laminin, fibronectin, type I, IV and V collagen and in the different spatial organization of matrix in type I collagen gels in three-dimensional culture (3D). In 2D culture PDGF BB was a potent mitogen, AB elicited an intermediate response while AA had no effect on cell proliferation. Extracellular matrix did not modify the PDGF responsiveness in 2D-culture. The different effects of the three PDGF isoforms were due to differential expression and isoform specific association of the PDGF-receptor subunits. Specifically, the beta-receptor was strongly expressed, whereas the alpha-receptor was only barely detectable on the cell surface. Metabolic labeling revealed synthesis and intracellular accumulation of the complete alpha-receptor protein, and treatment with suramin increased its surface expression, suggesting continuous receptor down-regulation by endogenous PDGF. Morphological and ultrastructural analysis in 3D culture revealed a change in mesangial cell phenotype, forming a branching network of multicellular structures. Assessment of proliferation in 3D culture showed quiescent cells and PDGF unresponsiveness. Investigation of the PDGF beta-receptors revealed a rapid down-regulation in 3D culture; both receptor subunits were not detectable on the cell surface. We conclude that 3D culture promotes the induction of a different mesangial cell phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Platelet-derived growth factor in renal development and disease.

In aggregate, the evidence reviewed here supports a very important role for PDGF expression and action at local glomerular and interstitial sites in human kidney development and disease. PDGF delivered by platelets, or produced by endogenous cells of the kidney is capable of stimulating responses including proliferation, matrix deposition, chemotaxis, and contraction in renal cells, particularly mesangial cells and interstitial fibroblasts. During kidney development, PDGF may mediate processes of cellular recruitment, extracellular matrix deposition, and proliferation with the constructive outcome of glomerulogenesis and vascularization. Proliferation and production of extracellular matrix components by these cells likely contribute to destructive proliferative and sclerotic responses attending proliferative and other glomerulopathies. As additional information accumulates, therapeutic targets within the PDGF system may provide opportunities to arrest destructive renal processes.

Animals↗
PubMed1991

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.

Animals↗
PubMed1991

Phospholipase C gamma complexes with ligand-activated platelet-derived growth factor receptors. An intermediate implicated in phospholipase activation.

Several steps implicated in platelet-derived growth factor (PDGF) receptor-coupled signaling are activated by PDGF exposure at 0-4 degrees C. These include receptor self-phosphorylation, physical association with and phosphorylation of phospholipase C gamma (PLC gamma). Reduced temperature blocks PDGF internalization, making it possible to dissociate bound PDGF after PLC gamma tyrosine phosphorylation. We addressed the functional consequences of PDGF dissociation from intact cell PDGF receptors. PDGF exposure at 0-4 degrees C for 15 min stimulated self-phosphorylation of a subpopulation of BALB/c 3T3 cell PDGF beta-type receptors (35%) and initiated subsequent inositol phosphate production. A small fraction of cellular PLC gamma (1-3%) coprecipitated with ligand-activated PDGF receptors; 3-5% of cellular PLC gamma acquired phosphotyrosine. The PLC gamma coprecipitating with PDGF receptors did not contain detectible phosphotyrosine. Phosphotyrosine antibody recovered similar amounts of PLC gamma from soluble and particulate fractions of PDGF-stimulated cells. Acid dissociation of bound PDGF from receptor caused rapid dephosphorylation of PDGF receptors and PCL gamma, and interrupted PLC gamma-PDGF receptor coprecipitation. Orthovanadate blocked tyrosine dephosphorylation of both PDGF receptors and PLC gamma and stabilized coprecipitation. Orthovanadate reversed the acid wash effect to abrogate PDGF-stimulated inositol phosphate production. PDGF receptor remains competent to coprecipitate with PLC gamma and stimulate PLC-mediated inositol phosphate production if PDGF-induced receptor phosphorylation is maintained. Formation of a coprecipitable PDGF receptor-PLC gamma complex appears required for PDGF-stimulated inositol phosphate production.

Blotting, Western↗
PubMed1991

5' untranslated sequences determine degradative pathway for alternate PDGF B/c-sis mRNA's.

The 5' untranslated sequence (5' UTS) of the platelet-derived growth factor B (PDGF B/c-sis) mRNA is GC rich, includes AUGs upstream of the translation initiation site, and inhibits translation of downstream heterologous and autologous coding sequences. Its primary sequence has been remarkably conserved through evolution. In this study, we identified two additional PDGF B/c-sis mRNAs expressed in endothelial cells. These were shown by Northern, primer extension, and nuclease protection analyses to differ in extent of 5' untranslated first exon sequence. The predominant of these 5' truncated transcripts was 2.8kb, extended 15nt upstream of the translation start site and was markedly stabilized by cycloheximide and anisomycin, but not puromycin or pactamycin. Cycloheximide increased the half-life of this mRNA approximately 7 fold, without affecting stability of the full length mRNA. Cycloheximide withdrawal caused its abrupt destabilization. The 2.8kb PDGF B/c-sis mRNA lacks translation inhibitory 5' UTS, encodes the same PDGF B protein, appears initiated from within first exon genomic sequences, and is degraded through a process that is sensitive to translation arrest and distinct from that degrading the 3.8kb PDGF B/c-sis mRNA.

Anisomycin↗
PubMed1989

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator.

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.

Cells, Cultured↗
PubMed1989

Epidermal growth factor binding, stimulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule.

Epidermal growth factor and insulin share many biological activities, including stimulation of cell proliferation, ion flux, glycolysis, fatty acid and glycogen synthesis, and activation of receptor-linked tyrosine kinase activity. In the kidney, insulin has been shown to regulate transport processes and inhibit gluconeogenesis in the proximal tubule. Since the kidney represents a major source of EGF, the present studies investigated whether proximal tubule contained EGF receptors, whether EGF receptors were localized to apical or basolateral membranes, and whether EGF receptor activation participated in the regulation of an important proximal tubule function, gluconeogenesis. Specific EGF receptors were demonstrated in the basolateral membrane of proximal tubule. Following incubation with 125I EGF, basolateral membranes demonstrated equilibrium binding at 4 degrees C and 23 degrees C. There was 78 +/- 2% specific binding (n = 13). The dissociation constant (Kd) was 1.5 x 10(-9) M and maximal binding was 44 fmol/mg protein. There was ninefold more specific binding to proximal tubule basolateral membrane than to brush border membrane. In basolateral, but not brush border membranes, EGF induced phosphorylation of the tyrosine residues of intrinsic membrane proteins, including a 170 kDa protein, corresponding to the EGF receptor. In the presence of the gluconeogenic substrates, alanine, lactate, and succinate, proximal tubule suspensions synthesized glucose. EGF inhibited glucose production in a concentration-dependent manner over a concentration range of 3 x 10(-11) to 3 x 10(-9) M. In addition, EGF inhibited angiotensin II-stimulated glucose production in the proximal tubule suspensions. EGF did not significantly increase net glucose metabolism nor decrease cellular ATP concentrations. Therefore, these studies demonstrated that rat proximal tubule contained specific receptors for EGF that were localized to the basolateral membrane and linked to tyrosine kinase activity. EGF significantly inhibited proximal tubule glucose production without significantly increasing net glucose consumption.

Animals↗
PubMed1989

Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C.

Phospholipase C (PLC)-mediated production of inositol trisphosphate and diacylglycerol is stimulated by binding of platelet-derived growth factor (PDGF) to cell-surface receptors. Antibodies recognizing native PDGF receptors through peptide-domain epitopes coprecipitated 4-fold more PLC activity with receptors from PDGF-stimulated than from unstimulated BALB/c 3T3 cells, despite equivalent PDGF receptor recovery. Activity coprecipitated from unstimulated cells was comparable to nonspecific activity recovered with preimmune sera or in the presence of competing peptide immunogen. PLC activity coprecipitating with PDGF receptors represented 60% of anti-phosphotyrosine antibody-recovered activity from PDGF-stimulated cells. Coprecipitation was rapidly induced in cells treated with PDGF at 4 degrees C, reversibly lost with acid dissociation of PDGF from receptors, and recovered with PDGF readdition. PDGF concentrations effecting coprecipitation correlated with stimulation of intact-cell inositol phosphate production. Monoclonal antibodies to PLC gamma (145 kDa) coprecipitated from PDGF-stimulated cell lysates (but not from unstimulated cell lysates) tyrosine kinase activity that phosphorylated PDGF receptor and PLC gamma. Stable physical association of PDGF receptors with PLC may participate in coupling ligand binding to increased PLC activity.

Animals↗
PubMed1988

Distinct pathways mediate transcriptional regulation of platelet-derived growth factor B/c-sis expression.

The biochemical mechanisms responsible for regulating cellular platelet-derived growth factor expression are incompletely understood. Our previous studies have shown that platelet-derived growth factor B/c-sis mRNA levels are induced in human renal microvascular endothelial cells by either thrombin or transforming growth factor (TGF-beta), while exposure to agents which elevate cAMP levels blocks the induction responses. The current studies use combined transcription run-off and message decay rate experiments to show greater than 3-fold increases in rate of transcription after stimulation with either thrombin or TGF-beta. c-sis message has a 70-90-min half-life under basal conditions that is effectively unaltered by thrombin or TGF-beta. Forskolin does not decrease the stability of c-sis mRNA, although it attenuates transcription increases seen with inducing agents. TGF-beta induction of c-sis transcription is mediated independent of the protein kinase C (Ca2+- and phospholipid-dependent enzyme)-mediated responses to phorbol ester, as it remains intact following down-regulation of protein kinase C response; TGF-beta and phorbol elicit additive induction. Inhibitory effects of cAMP upon transcription act distal to early thrombin-receptor-coupled increases in phosphatidylinositol turnover and are capable of turning off TGF-beta-activated transcription after activation has been established. Both inducing and suppressing agents alter endothelial platelet-derived growth factor B/c-sis mRNA expression dominantly through effects upon rates of transcription, cAMP suppression of transcription is dominant, and TGF-beta and phorbol esters mediate induction of transcription through distinct pathways.

Cell Nucleus↗
PubMed1987

Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta.

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin. We now show that another agent of possible importance in mediating proliferation of cells adjacent to the endothelial cell layer, transforming growth factor-beta (TGF-beta), also induced c-sis expression in these cells. In addition, we have studied the effect of agents that increase intracellular cAMP levels upon the induction of endothelial cell c-sis mRNA. The adrenergic agonists isoproterenol and norepinephrine blocked the elevation of cellular c-sis mRNA accompanying exposure to either thrombin or TGF-beta. This effect was mediated through beta-adrenergic receptors, since propranolol but not phentolamine reversed the inhibition. Forskolin, a direct activator of adenylate cyclase, also blocked induction of c-sis mRNA by thrombin and TGF-beta and inhibited the release of PDGF activity into the media of these cells. Basal, as well as stimulated c-sis mRNA levels were attenuated by these agents that increase cellular cAMP levels. These data suggest that increased cAMP production inhibits the expression of c-sis encoded mitogens by endothelial cells, and that c-sis expression is subject to bidirectional regulation in these cells.

Animals↗
PubMed1987

Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors.

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.

Animals↗
PubMed1987

Cyclosporin A inhibits kinase C-independent activation of the Na+/H+ exchanger by PDGF and vanadate.

Mitogen-induced activation of Na+/H+ exchange was studied in Swiss 3T3 fibroblasts. Phorbol myristic acetate (PMA) caused amiloride inhibitable cell alkalinization. PDGF and vanadate, but not bombesin or thrombin, caused additional alkalinization when given 10 min after a maximal dose of PMA. Down-regulation of kinase C by 24 hr PMA exposure prevented the alkalinization response to bombesin and thrombin, but not to PDGF or vanadate. Cyclosporin A specifically blocked the additional alkalinization after PDGF or vanadate in cells acutely exposed to PMA and in kinase C down-regulated cells. Thus, there are at least two independent pathways which activate Na+/H+ exchange. PMA, bombesin, and thrombin act via kinase C. PDGF and vanadate cause additional stimulation of the Na+/H+ exchanger by a kinase C-independent pathway, inhibitable by cyclosporin A.

Animals↗
PubMed1987

Interrelationship between growth factor-induced pH changes and intracellular Ca2+.

Many mitogens cause rapid changes in intracellular pH and Ca2+. We studied the patterns of pH and Ca2+ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca2+ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. PDGF and bombesin caused a rapid (maximum change, less than 2 min) cytoplasmic acidification of 0.03 pH unit followed by a slower (5-10 min) alkalinization of approximately 0.11 pH unit above the resting pH of 6.88. PMA caused alkalinization without causing the early acidification. Bradykinin caused rapid acidification without the slower net alkalinization. Ionomycin also caused acidification without subsequent alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca2+ response were also determined for each agent. PDGF and bombesin caused a transient increase in cytoplasmic Ca2+ from a resting level of 85 +/- 12 nM to 190 +/- 12 nM within 2 min and return to baseline within 5 min. PMA caused no change in intracellular Ca2+. Bradykinin caused the most rapid (maximum response, less than 20 sec) increase in intracellular Ca2+. For each agonist, the Ca2+ transient could be blocked by buffering intracellular Ca2+ with quin-2. In Ca2+-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. We propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca2+. However, growth factor-induced alkalinization via the Na+/H+ exchanger is independent of changes in Ca2+.

Animals↗
PubMed1986

Thrombin stimulates c-sis gene expression in microvascular endothelial cells.

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media. The thrombin effect was not mediated through the proteolytic activity of thrombin, as proteolytically inactive thrombin stimulated the c-sis expression as well as native thrombin. This stimulation was mimicked by exposure of cells to biologically active phorbol esters, suggesting that thrombin action may be mediated through activation of kinase C (Ca2+/phospholipid-dependent enzyme). Thus, thrombin regulates the expression and release of PDGF activity from endothelial cells in culture and may act in vivo to stimulate mitogen release from endothelial cells, thereby inducing proliferation of perivascular cells.

Angiotensin II↗
PubMed1985

Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody.

The platelet-derived growth factor (PDGF) receptor is a 180-kDa membrane glycoprotein. A protein of identical size, lectin affinity, and isoelectric point has been identified as a major substrate for PDGF-activated tyrosine kinase in stimulated 3T3 cells. We have purified this tyrosine-phosphorylated protein to homogeneity by using anti-phosphotyrosine immunoaffinity and lectin affinity steps. Demonstration that this purified tyrosine phosphoprotein is the PDGF receptor necessitated development of an assay capable of identifying specific 125I-labeled PDGF binding activity in soluble receptor preparations. PDGF receptor solubilized from 3T3 cell membranes with the detergent octyl beta-D-glucoside was precipitated on an artificial liposome matrix after receptor aggregation with concanavalin A. Precipitated binding sites display affinity and kinetic characteristics of PDGF receptors in cells and membranes. Preparations of the 180-kDa phosphoprotein that are greater than 90% homogeneous by silver stain and by [35S]methionine protein autoradiography have specific high affinity 125I-labeled PDGF binding sites (equilibrium dissociation constant, 0.1 X 10(-9) M). Binding activity enrichment in this preparation reflects an 11,000-fold purification of binding activity in intact cells. These data demonstrate that the 180-kDa substrate of the PDGF-stimulated tyrosine kinase is the PDGF receptor. Furthermore, these methods provide a means of purifying this and other tyrosine kinase substrates from growth factor-stimulated cells.

Animals↗
PubMed1983

Visualization of lipoprotein receptors by ligand blotting.

This paper describes the visualization of the low density lipoprotein (LDL) receptor by ligand blotting. Preparations of detergent-solubilized membranes are subjected to one- or two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, after which the proteins are transferred to nitrocellulose paper. The paper is incubated with native LDL and then with an 125I-labeled antibody against LDL, and the bound antibody is visualized by autoradiography. The success of LDL blotting depends on the omission of sulfhydryl reducing agents from the electrophoresis system. Intrachain disulfide bonds allow the receptor to retain its binding activity even after electrophoresis in the presence of SDS. In identifying LDL receptors, the ligand blotting technique is as sensitive as immunoblotting with a monoclonal antibody against the LDL receptor; it can therefore be used to identify receptors when no anti-receptor antibodies are available. We use this technique to show that the LDL receptor of the rabbit adrenal gland has the same molecular weight as the LDL receptor of the bovine adrenal cortex and human fibroblasts. The ligand blotting technique may be generally applicable for visualization of other plasma membrane receptors after SDS-gel electrophoresis.

Adrenal Glands↗