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Query: UNIPROT:P06889 (Mol)
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The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.
Mol Cell Biol 1990 Nov
PMID:Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation. 217 88

The hormonal regulation of the human urokinase type plasminogen activator (uPA) gene has been studied by introducing into mouse and rat Sertoli cell primary cultures a recombinant plasmid, in which the transcription regulatory elements of the cloned human uPA gene drive the expression of the bacterial chloramphenicol-acetyl-transferase gene. It was found to be expressed and regulated by FSH and (Bu)2cAMP in the mouse cells only, in agreement with data on the expression of the endogenous gene in rat and mouse gonads. The stimulation of transcription by FSH was evident in cultures from 13-day-old but not from 18-day-old mice, even though (Bu)2cAMP induction could be observed at both ages. Phorbol-myristate acetate was found to activate the human uPA promoter in Sertoli cell cultures from mice of both ages, even though the effect was less evident in cultures of 18-day-old animals. Deletion analysis of the human uPA 5'-flanking region showed that the distal enhancer element is not needed for (Bu)2cAMP induction, and that at least two promoter regions are involved in (Bu)2cAMP induced transcription. One of these cAMP responsive regions lies between nucleotides -72 and -29 from the CAP site. The sequence of this region would suggest the binding of transcription factor AP-2, a cell-specific mediator of both cAMP and phorbol esters action on gene expression. However, these sequences do not mediate phorbol ester activation of human uPA promoter in mouse Sertoli cells.
Mol Endocrinol 1990 Jun
PMID:Follicle-stimulating hormone and cyclic AMP induce transcription from the human urokinase promoter in primary cultures of mouse Sertoli cells. 217 96

Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t-PA) was expressed, N-glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214-223, 1989). We also showed that t-PA secretion was blocked by tunicamycin (TM), an inhibitor of N-glycosylation, but not by castanospermine (CS) or N-methyldeoxynojirimycin, inhibitors of the initial steps in N-linked oligosaccharide processing. This suggested that the addition, but not the processing, of N-linked oligosaccharides is required for the secretion of recombinant t-PA from baculovirus-infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N-glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane-bound glycoproteins were expressed in control, TM-treated, or CS-treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N-glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM-treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM-treated Sf9 cells by binding to them and producing transport-incompetent heterooligomeric complexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose-regulated 78 kilodalton protein (BiP/GRP78).
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PMID:Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells. 234 87

Calcitonin-induced changes in gene expression at the level of protein synthesis were examined in cultured porcine kidney cells (LLC-PK1) using two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides. Twelve intracellular polypeptides showed reproducible changes in the relative intensity of labeling. From that set of polypeptides ten showed an increase and two a decrease in the labeling intensity after calcitonin treatment. One of the induced polypeptides was identified as plasminogen activator and two others were shown to be related to cytokeratins. The labeling of two induced and well-separated polypeptides of 70K and 95K Mr was quantitated and correlated with an increase in secretion of plasminogen activator.
Mol Cell Endocrinol 1985 May
PMID:Hormonal regulation of protein synthesis in cultured kidney cells. 240 39

HTC rat hepatoma cells synthesize and secrete both tissue-type plasminogen activator (tPA) and type 1 plasminogen activator-inhibitor (PAI-1). Incubation with the synthetic glucocorticoid dexamethasone causes a rapid decrease in tPA activity which is secondary to a 5-fold increase in PAI-1 antigen and activity. Paradoxically, dexamethasone increases tPA antigen by 50%. We have analyzed HTC cell RNA by Northern and slot blot analysis, using as probes radiolabeled human PAI-1 and rat tPA cDNAs. HTC cells have a single species of PAI-1 mRNA of approximately 3.2 kilobases, which is increased 4-fold upon incubation with dexamethasone. Maximal induction occurs after 8-10 h of incubation. Half-maximal induction occurs at 5 nM dexamethasone. Dexamethasone also transiently increases the 2.8 kilobase tPA mRNA. The protein synthesis inhibitor cycloheximide does not affect accumulation of PAI-1 mRNA and does not block its induction by dexamethasone. In contrast, cycloheximide alone causes an increase in tPA mRNA, and in combination with dexamethasone, no further increase is observed. Induction of both mRNAs is prevented by actinomycin D. We conclude that the dexamethasone-induced increase in HTC cell PAI-1 activity and antigen is the result of a direct effect on accumulation of PAI-1 mRNA.
Mol Endocrinol 1989 Feb
PMID:Glucocorticoid induction of plasminogen activator and plasminogen activator-inhibitor messenger RNA in rat hepatoma cells. 246 9

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.
Mol Cell Biol 1989 Jan
PMID:Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. 249 30

Tumor necrosis factor-alpha (TNF-alpha) was found to induce type-1 plasminogen activator inhibitor (PAI-1) antigen in the human fibrosarcoma cell line HT-1080, and PAI-1 and urokinase-type plasminogen activator (u-PA) antigens in the human carcinoma cell line T-CAR1; tissue-type plasminogen activator (t-PA) antigen was not affected or slightly decreased. The effects in HT-1080 and T-CAR1 cells were preceded by increases in the cellular levels of the corresponding mRNAs. Cycloheximide caused an increase of PAI-1 mRNA in T-CAR1 cells, but not in HT-1080 cells; during this increase the relative abundance of the two PAI-1 mRNA species, of 2.3 kb and 3.4 kb, respectively, changed strongly in favor of the longer transcript. We conclude that TNF-alpha may affect proteolytic activity in the microenvironment of cells in malignant tumors by affecting gene expression of u-PA and PAI-1.
Mol Cell Endocrinol 1989 Jan
PMID:Tumor necrosis factor-alpha regulates mRNA for urokinase-type plasminogen activator and type-1 plasminogen activator inhibitor in human neoplastic cell lines. 250 Nov 20

The phenotype of a differentiated cell results from the expression of a unique set of genes in that cell. The differentiation of F9 teratocarcinoma cells in response to retinoic acid and cyclic AMP is an excellent example of this process, as the appearance of several gene products during the course of the differentiation process has been documented. In principle, the activation of gene expression could be due to the appearance of positive-acting factors, the loss of negative-acting factors, or a combination of both. Since F9 cells have been shown to express a cellular E1A analog whereas differentiated F9 cells do not, and it is known that the viral E1A gene exerts a negative effect on transcription of both viral and cellular genes, we determined whether the cellular genes activated during F9 cell differentiation are subject to E1A negative control. We found that infection of differentiated F9 cells with wild-type adenovirus resulted in a decline in the levels of collagen type IV mRNA and plasminogen activator mRNA, both of which are induced by differentiation. At least for the collagen gene, this phenomenon appears to involve a transcriptional repression.
Mol Cell Biol 1989 Jul
PMID:Adenovirus E1A-mediated negative control of genes activated during F9 differentiation. 252 83

Primary cultures of rat hepatocytes produce tissue-type plasminogen activator (tPA) and plasminogen activator-inhibitor type 1 (PAI-1). Incubation of hepatocytes with 50 microM 8-(4-chlorophenylthio)cAMP (CPT-cAMP) results in a 4-fold increase in tPA activity, whereas the synthetic glucocorticoid dexamethasone (1 microM) causes a more than 90% decrease. In combination, dexamethasone completely overcomes the CPT-cAMP effect and markedly decreases PA activity. PAI-1 is induced by both CPT-cAMP and dexamethasone, and the effects of these agents are additive. Accumulation of tPA mRNA is increased more than 4-fold by CPT-cAMP and is greatly decreased by incubation with dexamethasone. Dexamethasone in combination with CPT-cAMP totally blocks this cAMP effect. The protein synthesis inhibitor cycloheximide does not prevent either the dexamethasone-induced decrease or the CPT-cAMP-induced increase in tPA message and, in fact, augments the cAMP-induced increase in tPA mRNA. Hepatocyte PAI-1 mRNA levels are increased 2-fold by incubation with either CPT-cAMP or dexamethasone; in combination, these effectors cause a 4-fold increase in PAI-1 mRNA. Cycloheximide alone causes a marked increase in PAI-1 mRNA, but does not block the induction by either CPT-cAMP or dexamethasone. We conclude that incubation of hepatocytes with CPT-cAMP induces tPA activity by increasing tPA mRNA accumulation and that dexamethasone causes a decrease in tPA activity by both decreasing tPA mRNA and increasing PAI-1 mRNA and activity. Concomitant protein synthesis is not required for the regulation of tPA or PAI-1 mRNA by either CPT-cAMP or dexamethasone, indicating a primary effect of these agents on gene transcription or mRNA stability.
Mol Endocrinol 1989 Jan
PMID:Glucocorticoid and cyclic nucleotide regulation of plasminogen activator and plasminogen activator-inhibitor gene expression in primary cultures of rat hepatocytes. 253 89

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.
Mol Endocrinol 1989 Jan
PMID:Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium. 253 92


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