Birgitte Georg

Plasminogen activator inhibitors: hormonally regulated serpins

Etude de la regulation hormonale des enzymes appartenant a la famille de serine protease inhibiteur (plasminogen activator et urokinase)

Light and darkness regulate melanopsin in the retinal ganglion cells of the albino Wistar rat.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

Immunohistochemical Localization of the VIP1 Receptor (VPAC1R) in Rat Cerebral Blood Vessels: Relation to PACAP and VIP Containing Nerves

The two structurally related peptides, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), are present in cerebral vascular nerve fibers. Biologic actions of VIP are exerted through two receptors, VPAC1 and VPAC2, having similar binding affinity for both VIP and PACAP. In the current study, the authors have developed a specific antibody against the rVPAC1 receptor to examine the localization of rVPAC1 immunoreactivity in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence confocal microscopy. Specificity of the antiserum was ensured by immunoblotting and immunocytochemistry of cells transfected with cDNA encoding the different PACAP-VIP receptor subtypes. The rVPAC1 receptor immunoreactivity was localized to the plasmalemma of circularly orientated smooth muscle cells on superficial cerebral arteries and arterioles taken from the basal surface of the brain. By double immunostaining VIP immunoreactive nerve fibers and, to a lesser extent, those containing PACAP were shown to have intimate contact with the receptor protein. Vasoactive intestinal polypeptide and PACAP containing cerebrovascular nerve fibers were found in separate nerve populations with different distribution pattern and density. In brain sections processes of cortical VIP-, but not PACAP-, containing neurons seemed to innervate the rVPAC1 receptor of pial arterioles on the brain surface. The current findings provide the neuroanatomical substrate for a role of VIP and maybe PACAP in the regulation of cerebral blood flow.

Diurnal Rhythmicity of the Canonical Clock Genes Per1, Per2 and Bmal1 in the Rat Adrenal Gland is Unaltered after Hypophysectomy

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus (SCN), and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. Rhythmic expression of clock genes in the adrenal glands has previously been reported. Since the central clock in the SCN communicates with the adrenal glands via circadian release of adrenocorticotrophic hormone, we quantified the mRNAs for the canonical clock genes, Per1, Per2 and Bmal1 in the adrenal glands by real‐time reverse transcription‐polymerase chain reaction during a 24‐h‐cycle in normal and hypophysectomised rats. The mRNAs for all the three clock genes disclosed rhythmic oscillations with a period of 24 h and the phase did not differ between the hypophysectomised and intact rats. The expression pattern of Per1 and Bmal1 was in antiphase in both groups of animals. In situ hybridisation histochemistry using antisense RNA probes demonstrated that, at times of peak expression, mRNAs for all the three clock genes were expressed in the adrenal cortex with a particularly strong labelling in the zona reticularis. In accordance with the mRNA localisation, immunostaining for PER1 protein was visualised in cells of the adrenal cortex, being most intense in the inner zone. The immunostaining also demonstrated a translocation of PER1 protein from the cytoplasm to the nucleus during the daily cycle, supporting the existence of a core oscillator in the individual adrenal gland cells. Our findings support the existence of a circadian core oscillator in cells of the rat adrenal cortex and indicate that the activity of the oscillator is independent of SCN signalling via the pituitary gland. The adrenal cortical clock could be involved in rhythmic transcriptional activation of genes associated with hormonal biosynthesis, involved in gating of the response of the adrenal cortex to external cues or involved in apoptosis of adrenal cortical cells.

Plasminogen activator inhibitor type 1: cell-specific and differentiation-induced expression and regulation in human cell lines, as determined by enzyme-linked immunosorbent assay

We have performed a comparative study of the regulation by glucocorticoids and phorbol 12-myristate 13-acetate (PMA) of the production of type 1 plasminogen activator inhibitor (PAI-1) by 12 human cell lines. A sandwich-type enzyme-linked immunosorbent assay (ELISA) for PAI-1 that measures free PAI-1 as well as complexes between PAI-1 and both types of plasminogen activators has been used. Basal PAI-1 accumulation varied more than 5000-fold between the cell lines. No correlation was found between the PAI-1 level and other characteristics of the cell lines, except that three lines of SV40-transformed fibroblasts produced more PAI-1 than two non-transformed fibroblast cell lines. Three out of the 12 cell lines responded to glucocorticoids by an increased PAI-1 production. Four cell lines responded to PMA by an increased PAI-1 production. In addition, PMA-induced differentiation of the monocyte cell line U937 and the promyelocytic cell line HL-60 into macrophage-like cells was found to be correlated with an up to 100-fold increase in PAI-1 accumulation. The PMA-dependent differentiation of HL-60 cells led to acquisition of glucocorticoid inducibility of PAI-1. These findings provide information for future studies of the molecular mechanism of cell-specific expression and regulation of PAI-1.

Circadian Behaviour in Neuroglobin Deficient Mice

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

Tumor necrosis factor-α regulates mRNA for urokinase-type plasminogen activator and type-1 plasminogen activator inhibitor in human neoplastic cell lines

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.

Homer-1 mRNA in the rat suprachiasmatic nucleus is regulated differentially by the retinohypothalamic tract transmitters pituitary adenylate cyclase activating polypeptide and glutamate at time points where light phase-shifts the endogenous rhythm

The suprachiasmatic nucleus (SCN) generates circadian rhythms which are synchronised to the environmental light/dark cycle via the retinohypothalamic tract (RHT). Pituitary adenylate cyclase activating polypeptide (PACAP) and glutamate, two transmitters co-stored in the rat retinohypothalamic tract, are involved in photic entrainment of the circadian pacemaker, but their functional interplay is poorly understood. Homer proteins are involved in glutamatergic receptor function and signalling. By quantitative in situ hybridisation histochemistry we found that light stimulation of rats at early and late night induced Homer-1 gene expression in the SCN at time points where light induces phase-delay or phase-advance, respectively. Using a rat brain slice model Homer-1 mRNA levels in the SCN displayed a modest diurnal variation similar to that in vivo. The changes in Homer-1 gene expression after in vitro stimulation with PACAP and/or glutamate differed at early and late night. Nanomolar PACAP induced Homer-1 gene expression at both early and late night while glutamate was only able to increase Homer-1 mRNA level at early night. PACAP in micromolar concentration had no effect per se, but inhibited the glutamate induced Homer-1 response at early night, while at late night co-administration of PACAP and glutamate mediated a slight induction of Homer-1 gene expression. In conclusion, the RHT transmitters PACAP and glutamate could be responsible for the light-induced expression of Homer-1 in the SCN, and Homer-1 seems to be differentially regulated by the two transmitters at early and late night.

Expression of the clock genes Per1 and Bmal1 during follicle development in the rat ovary. Effects of gonadotropin stimulation and hypophysectomy

Daily oscillations of clock genes have recently been demonstrated in the ovaries of several species. Clock gene knockout or mutant mice demonstrate a variety of reproductive defects. Accumulating evidence suggests that these rhythms act to synchronise the expression of specific ovarian genes to hypothalamo-pituitary signals and that they are regulated by one or both of the gonadotropins. The aim of this study has been to examine the spatio-temporal expression of the clock genes Per1 and Bmal1 during gonadotropin-independent and gonadotropin-dependent follicle development in the rat ovary. We have examined the ovaries of prepubertal rats, of prepubertal rats stimulated with equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and of hypophysectomised adult animals. Using quantitative reverse transcription with the polymerase chain reaction, in situ hybridisation histochemistry and immunohistochemistry, we have demonstrated that the expression of the two clock genes is low and arrhythmic in ovarian cells during early gonadotropin-independent follicle development in prepubertal animals and in hypophysectomised animals. We have also demonstrated that the expression of the clock genes becomes rhythmic following eCG stimulation in the theca interna cells and the secondary interstitial cells and that, following additional hCG stimulation, the expression of the clock genes also becomes rhythmic in the granulosa cells of preovulatory follicles. These findings link the initiation of clock gene rhythms in the rat ovary to the luteinising hormone receptor and suggest a functional link to androgen and progesterone production. In hypophysectomised animals, rhythmic clock gene expression is also observed in the corpora lutea and in secondary interstitial cells demonstrating that, in these compartments, entrainment of clock gene rhythms is gonadotropin-independent.

Chamber-dependent circadian expression of cardiac natriuretic peptides.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have important local functions within the myocardium, where they protect against accelerated fibrosis. As circadian expression of cardiac natriuretic peptides could be of importance in local cardiac protection against disease, we examined the diurnal changes of the mRNAs encoding ANP, BNP, and their common receptor NPR-A in atrial and ventricular myocardium. Forty eight mice were killed at the following ZT times: 4, 8, 12, 16, 20, and 24, where ZT designates Zeitgeber; ZT 0 corresponds to lights ON and ZT 12 corresponds to lights OFF. Eight animals (4 males and 4 females) were included at each time point. Another 48 animals were killed during the second cycle of dark/dark (designated Circadian Time or CT: CT 4, CT 8, CT 12, CT 16, CT 20, and CT 24). The cellular contents of the clock genes Per1 and Bmal1 as well as ANP, BNP, and their common receptor (NPR-A) were determined using RT-PCR. Per1 and Bmal1 mRNA contents oscillated in antiphase in both atrial and ventricular regions, where Bmal1 mRNA peaked 12h out of phase relative to Per1 mRNA. ANP and NPR-A atrial mRNA contents revealed borderline significant diurnal changes, whereas ventricular BNP mRNA contents exhibited pronounced oscillation during constant darkness with nadir at CT 12 (P<0.0001). In conclusion, we report a chamber-dependent circadian profile of cardiac BNP mRNA contents, which is not paralleled by the related ANP gene. Our findings suggest that the BNP mRNA pattern could be associated with increased cardiac susceptibility and response to disease.