Spontaneous caloric restriction associated with increased leptin levels in obesity-resistant αMUPA mice.

BACKGROUND: αMUPA mice carry as a transgene the cDNA encoding urokinase-type plasminogen activator, a member of the plasminogen/plasmin system that functions in fibrinolysis and extracellular proteolysis. These mice spontaneously consume less food when fed ad libitum and live longer compared with wild-type (WT) control mice. αMUPA mice are obesity resistant and they share many similarities with calorically restricted animals. However, extensive metabolic characterization of this unique transgenic model has never been performed. METHOD: Metabolism of αMUPA mice was analyzed by measuring hormone, lipid and glucose levels in the serum, as well as gene and protein expression levels in the liver, hypothalamus and brainstem. RESULTS: αMUPA mice were found to be leaner than WT mice mainly because of reduced fat depots. Serum analyses showed that αMUPA mice have high levels of the anorexigenic hormones insulin and leptin, and low levels of the orexigenic hormone ghrelin. Analyses of brain neuropeptides showed that the transcript of the anorexigenic neuropeptide Pomc is highly expressed in the brainstem, whereas the expression of the orexigenic neuropeptides Npy, Orexin and Mch is blunted in the hypothalamus of αMUPA mice. In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) levels were higher in the liver and lower in the hypothalamus, thus promoting simultaneously central reduction in appetite and peripheral loss of fat. The levels of SIRT1 were low in the liver, but high in the hypothalamus, a feature that αMUPA mice share with calorically restricted animals. CONCLUSION: Taken together, αMUPA mice exhibit a unique metabolic phenotype of low-calorie intake and high leptin levels, and could serve as a model for both spontaneous calorie restriction and resistance to obesity.

The suprachiasmatic nuclei are involved in determining circadian rhythms during restricted feeding.

The circadian clock in the suprachiasmatic nuclei (SCN) responds to light and regulates peripheral circadian rhythms. Feeding regimens also reset the clock, so that time-restricted feeding (RF) dictates rhythms in peripheral tissues, whereas calorie restriction (CR) affects the SCN clock. To better understand the influence of RF vs. CR on circadian rhythms, we took advantage of the transgenic alphaMUPA mice that exhibit spontaneously reduced eating, and can serve as a model for CR under ad libitum feeding, and a model for temporal CR under RF compared with wild type (WT) mice. Our results show that RF advanced and generally increased the amplitude of clock gene expression in the liver under LD in both mouse types. However, under disruptive light conditions, RF resulted in a different clock gene phase in WT mice compared with alphaMUPA mice, suggesting a role for the reduced calories in resetting the SCN that led to the change of phase in alphaMUPA mice. Comparison of the RF regimen in the two lighting conditions in WT mice revealed that mPer1, mClock, and mBmal1 increased, whereas mPer2 decreased in amplitude under ultradian light in WT mice, suggesting a role for the SCN in determining clock gene expression in the periphery during RF. In summary, herein we reinforce a role for calorie restriction in resetting the SCN clock, and unravel a role for the SCN in determining peripheral rhythms under RF.

A RUNX/AML-binding motif residing in a novel 13-bp DNA palindrome may determine the expression of the proximal promoter of the human uPA gene.

Urokinase-type plasminogen activator (uPA) is a multifunctional extracellular serine protease implicated in different events including fibrinolysis, tissue remodeling, and hematopoiesis. The human uPA gene contains a major promoter region at around 2000 bp upstream from the transcription start site (+1), and a second regulatory region spanning nucleotides -90/+32 within the proximal promoter. Here, an inspection of this region revealed a novel 13-bp palindrome residing at position +8/+20. Interestingly, the palindrome contains the DNA consensus-binding hexamer for the RUNX/AML family of transcription factors that play a role in hematopoiesis, leukemia, and several developmental processes. Measuring the expression for promoter-reporter constructs after transfection revealed that deletion of the palindrome abrogated most of the proximal promoter activity in 293A cell. Additionally, electrophoretic mobility shift assays have shown that the palindrome could bind the RUNX1 component in nuclear extracts of myeloid cell lines exclusively through its RUNX motif. The palindrome was found in five additional human genes, two of which (MYH11 and MLLT1) have been linked to chromosomal rearrangements leading to leukemia. The data presented here have implicated, for the first time, RUNX/AML in the regulation of the uPA gene. The significance of the novel palindrome regarding gene regulation through the RUNX motif deserves further investigation.

Plasminogen activator inhibitor-1 in cardiovascular cells: rapid induction after injecting mice with kainate or adrenergic agents.

OBJECTIVES: Plasminogen activator inhibitor-1 (PAI-1) is a major anti-fibrinolytic glycoprotein thought to promote vascular diseases. Recently we have shown that systemically injecting mice with kainate, an analog of the principal brain excitatory neurotransmitter glutamate, immediately induced PAI-1 mRNA in brain vascular cells which are not known to contain glutamate receptors. Here we further investigated whether: (a) kainate also increases PAI-1 gene expression in the cardiac vascular bed; (b) subunits of kainate/AMPA receptors could be expressed in cardiac and brain vascular cells; and (c) PAI-1 mRNA could be similarly induced by agonists of adrenergic receptors that are candidates to act downstream in kainate-activated pathways. METHODS: We analyzed cardiac and brain cryosections for PAI-1 mRNA, as well as mRNAs encoding three receptor subunits, by in situ hybridization using 35S-labeled specific riboprobes. PAI-1 activity was tested in cardiac homogenates using one-phase reverse zymography. RESULTS: Prominent PAI-1 mRNA hybridization signals were induced in the vascular cells of the heart, and unexpectedly, also in cardiocytes, within 1-2 h after injection of kainate (i.p., 11-25 mg/kg body weight); the signals persisted for at least 8 h and disappeared after 24 h. In addition, PAI-1 activity increased ( approximately 5 fold) 2-10 h after the treatment. In contrast, mRNAs encoding the kainate/AMPA receptor subunits could not be detected. The adrenergic agents adrenaline (3.5 mg/kg) and isoproterenol (200 mg/kg) exerted kainate-like effects in cardiovascular cells. CONCLUSIONS: These results revealed, for the first time, that PAI-1 gene expression can be enhanced locally in the cardiovascular system by a fast-acting neurological mechanism triggered by glutamate receptors, whose pathway and relation to catecholamines, which exerted similar effects, have yet to be resolved. These findings raised the possibility that excessive glutamate, or stress-related catecholamines, may increase the risk of stroke and myocardial infarction.

Plasminogen.

Plasminogen activator inhibitor-2 (PAI-2) specifically inhibits plasminogen activators, extracellular fibrinolytic serine proteases that are also implicated in brain plasticity and toxicity. Primarily localized intracellularly, PAI-2 is thought to also counteract apoptosis mediated by a currently undefined intracellular protease. Here we localized PAI-2 mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate excitation. We found that in the normal brain PAI-2 mRNA was confined to an area within the accumbens nucleus shell. After kainate was injected (i.p.), PAI-2 mRNA was substantially and rapidly (within 2 h) induced in neuron-like cells primarily in layers II-III of the neocortex; the cingulate, piriform, entorhinal and perirhinal cortices; the olfactory bulb, nucleus and tubercle; in the accumbens nucleus, shell and core; throughout the caudate putamen and the amygdaloid complex; in the CA1 and CA3 areas of the hippocampus, and in the parasubiculum. These findings suggest that PAI-2 could play a role in the accumbens nucleus as well as in activity-related events associated with olfactory, striatal, and limbic structures.

Plasminogen activator inhibitor-1 mRNA is localized in the ciliary epithelium of the rodent eye.

PURPOSE: To identify in the adult and developing rodent eye cells expressing the gene encoding plasminogen activator inhibitor-1 (PAI-1), an important component of the fibrinolytic system. METHODS: PAI-1 mRNA was localized in cryostat thin eye sections via in situ hybridization analysis using specific 35S-labeled riboprobes. PAI-1 activity was tested in the aqueous humor using one-phase reverse zymography. RESULTS: In the adult eye, PAI-1 mRNA was detected exclusively in epithelial cells of the ciliary processes, primarily in the apexes. In addition, PAI-1 activity was detected in the aqueous humor. PAI-1 mRNA was first found in the ciliary epithelium in embryonic day 18.5, when the ciliary body has reached an advanced developmental stage. PAI-1 mRNA was also detected in the ganglion cell layer of the retina at postnatal days 1 to 4, when angiogenesis takes place. CONCLUSIONS: During development, PAI-1 is likely to be involved in retina vascularization, in agreement with other cases of angiogenesis. Results for the adult eye indicate that the ciliary epithelium is the source for PAI-1 activity found in the aqueous humor. The results suggest that PAI-1 plays a role in balancing fibrinolysis and proteolysis specifically in the anterior segment of the eye, implying that PAI-1 overproduction in the ciliary epithelium could shift the balance against proteolysis and thus may interfere with aqueous outflow.

AlphaMUPA mice: a transgenic model for increased life span.

AlphaMUPA is a line of transgenic mice that, compared with their wild type (WT) counterparts, spontaneously eat less (approximately 20%) and live longer (average approximately 20%), thus resembling dietary-restricted (DR) mice. Here, we show that body temperature was significantly reduced in alphaMUPA compared with WT throughout a wide range of ages. Plasma corticosterone was significantly higher in young alphaMUPA compared to young WT; however, it significantly declined in aged alphaMUPA, but not in aged WT. In addition, age-associated thymus involution occurred in alphaMUPA as it did in WT. Thus alphaMUPA mice appear to largely resemble, but also to somewhat differ from diet-restricted animals. We also report on four new transgenic lines that, like alphaMUPA, produced in the brain the mRNA that encodes the extracellular protease urokinase (uPA); however, transgenic uPA expression was most extensive and widespread in the alphaMUPA brain, where it also occurred in the hypothalamus. AlphaMUPA was also the only line that ate less, but also showed another characteristic, high frequency leg muscle tremor seen only at unstable body states. We hypothesize that transgenic uPA in the brain could have caused the alphaMUPA phenotypic alterations. Thus alphaMUPA offers a unique transgenic model of inherently reduced eating to investigate the homeostatic state of delayed aging at the systemic and single-cell levels.

The 3'-untranslated region of the urokinase gene enhances the expression of chimeric genes in cultured cells and correlates with specific brain expression in transgenic mice.

Transgenic mice were previously described, carrying the cDNA of the human or murine protease urokinase-type plasminogen activator (uPA) while linked to the cell-specific promoter of the alphaA-crystallin gene. Surprisingly, these mice produced transgenic uPA in an ectopic manner specifically in the brain. Here we tested the possibility that this ectopic expression could have been contributed primarily by the uPA transgenic moiety. Several experimental approaches have been used. (a) Constructs consisting of uPA cDNA linked to the cell-specific promoters of the alphaA-crystallin or insulin genes yielded active uPA after transfection into cells where these promoters are thought to be inactive. (b) When reporter genes were inserted into these constructs between the promoter and the cDNA, the cDNA enhanced the chimeric reporter expression 5-50-fold. This effect was obtained upon stable or transient construct transfection into four different cell types. (c) Reporter enhancement also took place in the presence of the homologous uPA gene promoter. (d) Mapping of the cDNA through deletion-substitution analysis has detected fragments mediating positive or negative effects on reporter expression, all fragments residing in the 3'-untranslated region (3'UTR) of the uPA gene that was included in the cDNA. Some fragments exhibited cell-specific effects. One fragment (2002/2187) behaved like a classical transcriptional enhancer, enhancing reporter expression from different positions and orientations. (e) Transgenic mice have now been generated that carry a transgene consisting of the alphaA-crystallin promoter, the luciferase reporter gene and mouse uPA cDNA. Among four transgenic lines producing luciferase activity in the eye lens, three lines exhibited ectopic luciferase activity exclusively in the brain, where luciferase mRNA was localized through in situ hybridization. From these results we conclude that the 3'UTR of the uPA gene contains sequences capable of exerting variable effects on gene expression, including transcriptional enhancement. In addition, uPA cDNA correlates with transgenic brain expression. Therefore, we suggest that the 3'UTR of the uPA gene is involved in brain expression of the transgenes containing uPA cDNA as well as of the normal uPA gene.

mRNAs encoding urokinase-type plasminogen activator and plasminogen activator inhibitor-1 are elevated in the mouse brain following kainate-mediated excitation.

Urokinase-type plasminogen activator (uPA) is an inducible extracellular serine protease implicated in fibrinolysis and in tissue remodeling. Recently, we have localized uPA mRNA strictly in limbic structures and the parietal cortex of the adult mouse brain. Here, we tested whether the systemic treatment of mice with kainic acid (KA), an amino acid inducing limbic seizures, could elevate in the brain mRNAs encoding uPA and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), a major antifibrinolytic agent. Brain sections encompassing the hippocampus were tested through in situ hybridization using radiolabeled riboprobes specific for the two mRNA species. The results showed that KA greatly enhanced both mRNA species in sites of limbic structures and cortex. However, in the hypothalamus and brain blood vessels only PAI-1 mRNA was elevated. Those were also the only two locations where PAI-1 mRNA was detected in the non-treated control brain, although at a low level. For both mRNAs, KA enhancement was first evident 2-4 h after treatment, and it was most prolonged in the hippocampal area, where prominent hybridization signals persisted for three days. Here, both mRNAs were initially elevated in the hilar region of the dentate gyrus and in the molecular and oriens layers; however, PAI-1 mRNA became evident throughout the area, while uPA mRNA became especially pronounced in the CA3/CA4 subfield. In the cortex both mRNA types were induced, but only uPA mRNA was elevated in the retrosplenial cortex, and also in the subiculum. In the amygdaloid complex, uPA mRNA was restricted to the basolateral nucleus, whereas PAI-1 mRNA was seen throughout the structure, however, excluding this nucleus. These data show that seizure activity enhances the expression of uPA and PAI-1 genes in the brain; the patterns of enhancement suggest that the protease and its inhibitor may act in brain plasticity in synchrony, however, also independently of each other. Furthermore, the results suggest that by elevating PAI-1 mRNA in brain blood vessels, limbic seizures generate a risk for stroke.

Transgenic mice overexpressing urokinase-type plasminogen activator in the brain exhibit reduced food consumption, body weight and size, and increased longevity.

Transgenic mice designated alpha MUPA overproduce in many brain sites the urokinase-type plasminogen activator (uPA), a protease implicated in fibrinolysis and extracellular proteolysis. Here we report that, compared to their parental wild-type control, alpha MUPA mice spontaneously consumed less food (approximately 20%), exhibited reduced body weight (approximately 20%) and length (approximately 6%), and also prolonged life span (approximately 20%). The alpha MUPA phenotype is thus reminiscent of experimental animals in which dietary restriction enhances longevity. Reduced eating and body weight were observed in alpha MUPA mice shortly after weaning, and these levels were maintained virtually throughout their lifetime. alpha MUPA mice also exhibited lower levels of blood sugar (approximately 9%), smaller litter size (approximately 14%), and lower birth frequency (approximately 10%). In the adult alpha MUPA brain, uPA mRNA has been localized through in situ hybridization also in neuronal cells of the hypothalamic paraventricular nucleus, a region implicated in feeding behavior. No signals of uPA mRNA could be detected in the paraventricular nucleus of control mice. It is suggested that in alpha MUPA mice, overproduction of uPA in brain sites controlling feeding leads to reduced food consumption that, in turn, results in retardation of growth and body weight and also in increased longevity. The alpha MUPA experimental model may have implications for normal mice.

Localization of urokinase-type plasminogen activator mRNA in the adult mouse brain.

Urokinase-type plasminogen activator (uPA) is an inducible serine protease, secreted by a variety of cell types, that functions in fibrinolysis and has been implicated also in events such as cell migration and tissue remodeling and repair. To explore the role of uPA in the adult brain we have now screened the whole mouse brain for cells expressing the uPA gene through in situ hybridization using 35S-complementary RNA. uPA mRNA was visualized predominantly in three regions: (1) the subicular complex, (2) the entorhinal cortex, (3) the parietal cortex, where the signal was somewhat lower and confined to layers IV and VI. Weaker signals were seen in the basolateral nucleus of the amygdala and in the anterodorsal thalamic nucleus, and also in the hilus of the dentate gyrus where labeling was slightly over background. Cells exhibiting uPA mRNA signaling were large neurons according to morphological criteria. These results support the view of uPA being involved in neuronal functions of the adult brain, specifically in the hippocampal formation and the parietal cortex.

Components of the plasminogen activator system in astrocytes are modulated by tumor necrosis factor-alpha and interleukin-1 beta through similar signal transduction pathways.

Migration of astrocytes is thought to play a role in nerve regeneration and to be mediated, at least in part, by inflammation-associated cytokines. Plasminogen activators are secreted proteases that function in fibrinolysis and participate in cellular migration and invasion and, in some cases, are modulated by cytokines. Here, we show that two cytokines, tumor necrosis factor-alpha and interleukin-1 beta, can modulate plasminogen activation in astrocytes, each causing 90% reduction of total plasminogen activator activity. Direct and reverse zymography indicated that this reduction resulted from two simultaneous events, a pronounced decrease in tissue-type plasminogen activator activity and an induction of plasminogen activator inhibitor-1. Northern hybridization analysis indicated a 30-fold increase of the steady-state level of plasminogen activator inhibitor-1 mRNA following treatment with each of the two cytokines. Both of the cytokine-induced effects could be blocked by cycloheximide or actinomycin D. When signal transduction pathways were blocked, the results indicated the involvement of reduction in cyclic AMP levels, protein kinase activity, and arachidonic metabolites of the lipoxygenase pathway. The results thus show that the two cytokines reduce the ability of astrocytes to conduct fibrinolysis and extracellular proteolysis, and suggest that the effect of these cytokines on members of the plasminogen activation system is through a common signal transduction pathway.

Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning.

Transgenic mice designated alpha MUPA overproduce in the brain murine urokinase-type plasminogen activator (uPA), an extracellular protease implicated in tissue remodeling. We have now localized, by in situ hybridization, extensive signal of uPA mRNA in the alpha MUPA cortex, hippocampus, and amygdala, sites that were not labeled in counterpart wild-type mice. Furthermore, biochemical measurements reveal a remarkably high level of enzymatic activity of uPA in the cortex and hippocampus of alpha MUPA compared with wild-type mice. We have used the alpha MUPA mice to examine whether the abnormal level of uPA in the cortex and the limbic system affects learning ability. We report that alpha MUPA mice perform poorly in tasks of spatial, olfactory, and taste-aversion learning, while displaying normal sensory and motor capabilities. Our results suggest that uPA is involved in neural processes subserving a variety of learning types.

Human and murine urokinase cDNAs linked to the murine alpha A-crystallin promoter exhibit lens and non-lens expression in transgenic mice.

cDNAs encoding either the human or the murine urokinase-type plasminogen activator (uPA) were fused downstream from the promoter-enhancer element of the murine gene encoding alpha A-crystallin, a protein found exclusively in the ocular lens. The DNAs were microinjected into fertilized mouse eggs as linear fragments free of bacterial sequences, and for each construct one line of transgenic mice was generated. In both lines transgenic uPA activity was detected in the ocular lens, in agreement with previous results reported on transgenic mice bearing genes fused to the same regulatory region. Unexpectedly however relatively high levels of this activity were found also in the retina, and furthermore, human uPA activity was found also in different parts of the brain and in the bone marrow, and to a lesser extent in the spleen, thymus and optic nerve. Transgenic uPA transcript was found in the lens, retina, brain and thymus of mice carrying the murine cDNA. Such a pattern of expression was different from that exhibited by the endogenous murine uPA gene and, excluding the lens, it appeared to be conferred by the cDNAs. The putative regulation by uPA cDNAs is suggested to be mediated through an internal enhancer-like element functioning in combination with the alpha A-crystallin promoter in a fashion independent of the specific nature of the promoter.

Differentiation and oncogenesis: phenotypically distinct lens tumors in transgenic mice.

We report on two lines of transgenic mice that express a murine alpha A-crystallin/SV40 tumor antigen fusion gene in the eye lens. The alpha T1 line develops fast growing, poorly differentiated lens tumors, whereas the alpha T2 line produces lens tumors that are slow growing and well differentiated. There is a striking difference between these two lines in the temporal and spatial patterns of tumor antigen expression during initial lens development. In the alpha T1 line, the transgene is expressed very early in development in most lens cells, and no primary fiber differentiation takes place. In the alpha T2 line, transgene expression occurs after primary fiber formation has been initiated, and is restricted to differentiating fiber cells. The anterior epithelium from both alpha T lines undergoes normal development and remains morphologically normal until after birth, although in alpha T1 mice, these anterior cells produce considerable amounts of SV40 tumor antigens. This suggests that the state of differentiation of the lens cell plays an important role in its response to oncogene products.

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells.

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Follicular fluid contents as predictors of success of in-vitro fertilization-embryo transfer.

In order to establish criteria for selection of the best ova in in-vitro fertilization-embryo transfer (IVF-ET) programes we have examined the follicular fluid (FF) levels of plasminogen activator (PA), collagenolytic activity, progesterone (P) and alpha 2 macroglobulin (alpha 2M) and related them to the success of pregnancy. PA activity was similar in FF of pregnant and nonpregnant cycles, 13.8 +/- 3.9 mU/ml versus 14.6 +/- 2.9 (mean +/- SEM) respectively. By contrast, FF from pregnant cycles exhibited lower collagenolytic activity (49.6 +/- 3.9% versus 67.9 +/- 3.0; P less than 0.001). Likewise, in a semi-quantitative assay of alpha 2M, only 18.4% of the aspirates from pregnant cycles showed a precipitation line, whereas 76.8% of those from non-pregnant cycles were positive. Levels of P in aspirates from pregnant cycles were in the intermediate range, as compared with those from non-pregnant cycles (0.06-5.5 micrograms/ml versus 0.02-12.0 micrograms/ml). All these assays can be completed before ET and performed in IVF-ET programmes. In conclusion, it seems that a combination of follicular alpha 2M levels and collagenolytic activity, and to a lesser extent addition of P assay, may serve as good criteria for selecting the best embryos for establishment of pregnancy.

Induction of urokinase-type plasminogen activator by UV light in human fetal fibroblasts is mediated through a UV-induced secreted protein.

Plasminogen activator was previously shown to be induced by UV light in human cells with low capacity to repair UV-induced DNA lesions. We now show that in human fetal fibroblasts UV light enhanced the two mRNA species coding for the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator, but immunological analysis revealed exclusively uPA activity. Several independent and complementary experiments indicated that induction of uPA was mediated, apparently entirely, through a UV-induced, secreted protein (UVIS) in the growth medium of irradiated cells. First, elevation of uPA mRNA after irradiation was severely blocked by cycloheximide. Second, replacement of conditioned medium in irradiated cells while the rate of plasminogen activator induction was maximal rapidly and completely stopped any further increase in uPA activity. Third, addition of the same removed conditioned medium to nonirradiated cells mimicked UV light in enhancing the level of uPA activity as well as that of uPA mRNA. Fourth, UVIS activity was completely lost by treating the conditioned medium with trypsin but not with nucleases. Kinetic measurements indicated that the accumulation of UVIS rather than the induction of uPA by UVIS conferred the rate-limiting step in the overall process of uPA induction. Both UV light and UVIS acted synergistically with inhibitors of DNA repair for uPA induction. Based on these results, a model is proposed implicating relaxation of DNA torsional stress of an as yet undefined DNA sequence(s) in the induction of UVIS, which is then responsible for activation of the uPA gene.

Intrafollicular distribution of plasminogen activators and their hormonal regulation in vitro.

Recent studies from our laboratory corroborated the suggested role of plasminogen activation in follicular rupture at ovulation, and its involvement in the activation process of collagenolysis in the follicle. In the present study, the molecular types and cellular source of plasminogen activator (PA) were examined. Explanted preovulatory follicles produced in vitro both urokinase type and tissue type (t-PA) activators. Upon gonadotropin stimulation a highly significant increase in t-PA, but not in urokinase type, was observed. Separation of the follicle into granulosa cells and residual tissue, mainly theca, revealed that both compartments produce both types of PA. The granulosa compartment was found to produce 80-90% of the total follicular PA activity. Gonadotropins stimulated predominantly t-PA. Most of the gonadotropin-enhanced PA activity produced by granulosa cells was secreted into the culture medium, whereas that from thecal origin remained in the tissue. Likewise, in whole follicles only about 10% of PA was secreted into the medium. Gonadotropin-induced PA activity in vitro was reduced by inhibitors of steroidogenesis. This inhibition was overcome by the addition of estradiol-17 beta. The inhibition of steroidogenesis affected predominantly the t-PA type of PA. In conclusion, the granulosa cells contribute most of the follicular PA activity, and t-PA is predominantly enhanced by gonadotropin and estrogen. It seems, therefore, that t-PA is the activator involved in the processes leading to follicular rupture.