Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta- responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of TGF beta on the secretion of PA activity by A549 cells. PA activity was quantitated by caseinolysis assays, and characterized by urokinase mRNA analysis, immunoprecipitation, and zymography assays. PA-inhibitor production was observed in autoradiograms of SDS-polyacrylamide gels and reverse zymography assays. It was found that TGF beta enhanced the production of PA activity by these cells, in accordance with an enhancement of urokinase mRNA levels. A concomitant stimulation of type 1 PA-inhibitor production was also observed in A549 cells in response to TGF beta. In contrast to the observations of A549 cells, TGF beta caused a decrease in the expression of both urokinase and the tissue-type PA mRNA in human embryonic WI-38 lung fibroblasts indicating opposite regulation of the expression of PAs in these cells. The results suggest that TGF beta may play a role in the regulation of the invasive, proteolytically active phenotype of certain lung carcinoma cells.     

Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: effects of epidermal growth factor on production of PAs in oocytes and cumulus cells.

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.     

Gastrin and epidermal growth factor induction of ornithine decarboxylase in rat colonic explants

An organ culture system was utilized to examine the effect of gastrin (G-17-I) and epidermal growth factor (EGF) on colonic mucosal ornithine decarboxylase (ODC) activity, and the expression of the ODC gene. Exposure of colonic mucosal explants to either gastrin or EGF (50-500 ng/ml) for only 4 h resulted in a profound stimulation (150-600%) in ODC activity over the basal level. These increases were essentially abolished by difluoromethylornithine (DFMO; 2 nmol/ml) or CaCl2 (2 umol/ml). Gastrin also activated the ODC gene in the colonic mucosa as evidenced by increased steady-state ODC mRNA levels in the colonic mucosal explants after 4 h exposure to the hormone, when compared with the controls. It is concluded that colonic mucosal ODC is responsive to both gastrin and EGF.     

EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

Background

Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation.

Methods

Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml) and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model.

Results

PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA) content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.     

Regulation of tissue-type plasminogen activator activity and messenger RNA levels by gonadotropin-releasing hormone in cultured rat granulosa cells and cumulus-oocyte complexes

Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels.     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

Overexpression of human bone morphogenetic protein receptor 2 does not ameliorate monocrotaline pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is associated with mutations of bone morphogenetic protein receptor 2 (BMPR2), and BMPR2 expression decreases with the development of experimental PAH. Decreased BMPR2 expression and impaired intracellular BMP signaling in pulmonary artery (PA) smooth muscle cells (PASMC) suppresses apoptosis and promotes proliferation, thereby contributing to the pathogenesis of PAH. We hypothesized that overexpression of BMPR2 in resistance PAs would ameliorate established monocrotaline PAH. Human BMPR2 was inserted into a serotype 5 adenovirus with a green fluorescent protein (GFP) reporter. Dose-dependent transgene expression was confirmed in PASMC using fluorescence microscopy, quantitative RT-PCR, and immunoblots. PAH was induced by injecting Sprague-Dawley rats with monocrotaline (60 mg/kg ip) or saline. On day 14, post-monocrotaline (MCT) rats received 5 x 10(9) plaque-forming units of either Ad-human BMPR2 (Ad-hBMPR2) or Ad-GFP. Transgene expression was confirmed by fluorescence microscopy, quantitative RT-PCR of whole lung samples, and laser-capture microdissected resistance PAs. Invasive hemodynamic and echocardiographic end points of pulmonary hypertension were assessed on day 24. Endogenous BMPR2 mRNA levels were greatest in resistance PAs, and expression declined with MCT PAH. Despite robust hBMPR2 expression in all lung lobes and within resistance PAs of treated rats, hBMPR2 did not lower mean PA pressure, pulmonary vascular resistance index, right ventricular hypertrophy, or remodeling of resistance PAs. Nebulized intratracheal adenoviral gene therapy with hBMPR2 reliably distributed hBMPR2 to resistance PAs but did not ameliorate PAH. Depressed BMPR2 expression may be a marker of PAH but is not central to the pathogenesis of this model of PAH.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Plasminogen activators in human breast cancer cell lines: hormonal regulation and properties

To understand the hormonal regulation of plasminogen activators (PAs) in human breast cancer, we have examined the hormonal regulation and properties of PAs in four human breast cancer cell lines that differ markedly in their estrogen receptor (ER) content: MCF-7 cells contain high levels of ER (approx 7 pmol/mg DNA) and their PA activity was increased 3-4-fold by physiological concentrations of estradiol; T47-D and ZR-75-1 cells contain lower levels of ER (0.9 and 2.1 pmol/mg DNA respectively) and their PA activity was also increased 3-4-fold by estradiol. In contrast, MDA-MB-231 cells, which do not contain ER, showed a high level of PA activity that was not modulated by estradiol. SDS-PAGE followed by zymography indicated that MCF-7 cells secreted tissue-type PA (t-PA), T47-D and ZR-75-1 cells secreted urokinase-type PA (u-PA), and MDA-MB-231 cells secreted both types of PAs. The types of PAs secreted by these cell lines did not change upon treatment with estradiol. Dose-response curves for the stimulation of MCF-7 PA activity by different estrogens showed an excellent correlation between affinities of the estrogens for ER and their potency in stimulating PA activity. With a clonal subline of MCF-7 cells, MCF-L, a soluble inhibitor of both t-PA and u-PA was secreted. Incubation of purified t-PA or u-PA with the serum-free conditioned medium from MCF-L cells resulted in a shift in the mobility of t-PA and u-PA in SDS-polyacrylamide gels to forms increased in molecular mass by about 50,000-70,000. The shifts in molecular mass could be prevented by the presence of the competitive inhibitor p-aminobenzamidine, indicating that the active sites of the PAs were involved in the formation of these complexes. Furthermore, co-cultivation, of RT4-D rat neuroblastoma cells, which exhibit high levels of t-PA activity, with MCF-L cells resulted in a marked decrease in the PA activity of the RT4-D cells. Our results were consistent with the following conclusions: t-PA, u-PA or both were secreted by human breast cancer cells. In the ER-containing cell lines, depending upon the specific cell line, t-PA or u-PA was stimulated by estrogens. The unstimulated levels of PA activity and the magnitude of PA stimulation by estrogens were not closely related to ER content.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B(12) (cobalamin) in the rat central nervous system.

We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B(12) (cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor-alpha (TNF-alpha)-mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl-deficient diet, might modify the levels of three physiological neurotrophic factors-epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)-in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl-deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl-deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivo study presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF-alpha genes in the CNS of TGX rats.     

Epidermal growth factor increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA turnover

Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3′ untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed.     

Antiulcerogenic potential of Strychnos potatorum Linn seeds on Aspirin plus pyloric ligation-induced ulcers in experimental rats

Strychnos potatorum (Fam: Loganiaceae) Linn seeds are useful in the treatment of gastropathy in Indian traditional system of medicine. The present study describes the antiulcerogenic potential of S. potatorum Linn seeds on aspirin plus pyloric ligation (Aspirin+PL)-induced gastric ulcer model to substantiate its folklore claim. The seed powder (SPP) and aqueous extract of the seeds (SPE) at two doses 100 and 200 mg/kg, p.o. prevented ulcer formation by decreasing acid secretory activity and increasing the mucin activity in rats. The antiulcerogenic potential was further confirmed by the histopathological studies of stomach mucosa. The results indicate that SPP and SPE exhibit antiulcerogenic activity by both antisecretory and mucoprotective actions. The mucoprotective action of SPP and SPE may be due to the presence of polysaccharides in seeds. The antiulcerogenic potential of SPP and SPE was compared with the standard antiulcer drug, ranitidine.     

Voltage-gated K(+)-channel activity in ovine pulmonary vasculature is developmentally regulated

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K(+) (K(v))-channel activity increases with maturation; and 3) O(2)-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O(2) tension, acute hypoxia induced a more rapid and greater change in [Ca(2+)](i) in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in K(v)-channel activity, the effects of the K(+)-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca(2+)](i). 4-AP (1 mM) caused PASMC [Ca(2+)](i) to increase by 94 +/- 22% in the fetus and 303 +/- 46% in the adult. K(v)-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O(2) sensing that may render the adult PASMCs more responsive to acute hypoxia.     

Phenolic and anthocyanin fractions from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration partially through RHOA and RAC1

The present study investigates the effect of anthocyanin (ACN), phenolic acid (PA) fractions, and their combination (ACNs:PAs) from wild blueberry powder (Vaccinum angustifolium) on the speed of endothelial cell migration, gene expression, and protein levels of RAC1 and RHOA associated with acute exposure to different concentrations of ACNs and PAs. Time-lapse videos were analyzed and endothelial cell speed was calculated. Treatment with ACNs at 60 μg/mL inhibited endothelial cell migration rate ( P ≤ 0.05) while treatment with PAs at 0.002 μg/mL ( P ≤ 0.0001), 60 μg/mL ( P ≤ 0.0001), and 120 μg/mL ( P ≤ 0.01) significantly increased endothelial cell migration rate compared with control. Moreover, exposure of HUVECs to ACNs:PAs at 8:8 μg/mL ( P ≤ 0.05) and 60:60 μg/mL increased ( P ≤ 0.001) endothelial cell migration. Gene expression of RAC1 and RHOA significantly increased 2 hours after exposure with all treatments. No effect of the above fractions was observed on the protein levels of RAC1 and RHOA. Findings suggest that endothelial cell migration is differentially modulated based on the type of blueberry extract (ACN or PA fraction) and is concentration-dependent. Future studies should determine the mechanism of the differential action of the above fractions on endothelial cell migration.     

Cytokines regulate plasminogen activation system in astrocytoma cells.

We report here that human astrocytoma cell line U373-MG is able to express genes of the following components of plasminogen activation system: PA1-1, PN-1, u-PA and t-PA. Treatment of these cells with IL-1beta results in accumulation of PA1-1, PN-1 and u-PA mRNAs, whereas t-PA mRNA remains unaffected. IFNy preferentially enhances PN-1 and PA1-1, EGF enhances PA1-1, u-PA and t-PA expression. Simultaneous addition of anti-inflammatory cytokines IL-4, IL-13 and IL-10 has little effect on the tested components, except induction of u-PA mRNA wich was further enhanced by IL-4. We have confirmed interesting time-dependent regulation of plasminogen activation system by EGF/IFNgamma. Cells stimulated with EGF/IFNgamma show at first increased proteolytic activity but after 24 h inhibition of proteolysis with PA1-1 would prevail. To understand the cooperative effect of EGF and IFNgamma in PA1-1 induction the kinetics of activation of STAT1 was studied. It was found that although EGF alone does not activate STAT1, the STAT1 binding activity in the cells treated with the mixture of EGF/IFNgamma was considerably prolonged. Our results indicate the importance of inflammatory cytokines and EGF in gene regulation of plasminogen activation system in astrocytoma cells.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Retinoid modulation of plasminogen activator production in rat Sertoli cells

Tissue type (t) and urokinase type (u) plasminogen activators (PAs) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon FSH stimulation or upon the presence of adjacent spermatogenic cells. In the present study we have analyzed the production of PAs by retinoid-treated rat Sertoli cells. In addition, because retinoids modulate the response of Sertoli cells to FSH either potentiating or antagonizing its action, we have investigated a possible modulation of FSH-stimulated PA production. Under basal conditions, Sertoli cells, isolated from prepubertal rats, secrete predominantly uPA. A significant dose-dependent inhibition of uPA activity was observed after treatment with retinol, while no significant effect was detected upon tPA secretion. When Sertoli cells were cultured in the presence of 0.25 microM retinol, a significant inhibition of uPA activity was evident after 16 h of treatment and reached approximately 80% after 48 h of treatment. The analysis of the mRNA levels revealed that retinol induces an inhibition of the steady-state levels of uPA mRNA without affecting those of tPA. Moreover, retinol affected uPA mRNA levels by increasing mRNA turnover. The effect of retinoids on Sertoli cells isolated from older animals was less evident, possibly due to the reduced production of uPA with the increase of age of the donor animals. Our results on the effect of retinoids upon Sertoli cell uPA production reinforce the importance of retinoids in the control of postnatal testis development.     

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages – regenerating tissue and differentiated shoots. We observed significant (P<0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P<0.05), and lower levels of Spm (P<0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P<0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P<0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.