Progesterone receptor regulation by retinoic acid in the human breast cancer cell line T-47D

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.     

Changes in expression of the neuropeptide Y Y1 receptor gene in the medial amygdala of transgenic mice during pregnancy and after delivery

Long-term administration of progesterone or allopregnanolone was previously shown to increase Y1 receptor gene expression in the medial amygdala of Y1R/LacZ transgenic mice, which harbor a construct comprising the murine Y1 receptor gene promoter and a lacZ reporter. We have now investigated the effects of physiological fluctuations in the cerebrocortical concentrations of neuroactive steroids during pregnancy on Y1R/LacZ transgene expression by quantitative histochemical analysis of beta-galactosidase activity. Cerebrocortical concentrations of progesterone and its metabolites allopregnanolone and allotetrahydrodeoxycorticosterone were increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Transgene expression in the medial amygdala was also increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Similar results were obtained after analysis of Y1R mRNA levels in the medial amygdala of pregnant mice by in situ hybridization. Administration of the 5alpha-reductase inhibitor finasteride to pregnant mice prevented both the increase in the cerebrocortical concentrations of neuroactive steroids as well as the increase in transgene expression. These data suggest that fluctuations in the brain concentrations of endogenous neuroactive steroids during pregnancy are associated with changes in Y1 receptor gene expression in the medial amygdala, further supporting a functional interaction between the GABAergic and NPY-Y1 receptor systems.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

Developmental expression of metabotropic P2Y(1) and P2Y(2) receptors in freshly isolated astrocytes from rat hippocampus

There are at least three subtypes of cloned metabotropic P2 receptors linked to intracellular Ca(2+) rises in rat brain cells, namely, P2Y(1), P2Y(2) and P2Y(4). In this study we explore the subtypes of the metabotropic P2 receptors seen in freshly isolated astrocytes (FIAs) from P8-P25 rats. We found by single cell RT-PCR that in process-bearing FIAs from hippocampi of P8-P12 rats, 31% of the glial fibrillary acidic protein (GFAP) mRNA (+) cells expressed P2Y(1) mRNA while only 5% of the cells tested expressed P2Y(2) mRNA. The expression of P2Y(1) receptor mRNA was not changed in FIAs from the hippocampi of P18-P25 rats, but 38% of the GFAP mRNA (+) cells in the P18-P25 age group then showed P2Y(2) mRNA. We also studied whether the mRNA was expressing functional receptor protein by measuring Ca(2+) responses to specific agonists for P2Y(1) and P2Y(2). We found that similar proportions of GFAP mRNA (+) FIAs responded to ATP or UTP as showed mRNAs for P2Y (1) and P2Y(2,) respectively. Total tissue RNA from P9 and P24 rat hippocampus showed a 2.8-fold increase in P2Y(2) mRNA levels from P9 to P24 with a decrease in P2Y(1) mRNA. Thus, this study shows a marked up-regulation of mRNA for P2Y(2) from 9 to 24 days in rat hippocampus, and some of this increase is likely due to the protoplasmic astrocytes which is being translated into functional receptor protein in these cells.     

Altered Function of Peripheral Organ Systems in Rats Exposed to Chronic Mild Stress Model of Depression

1. In depression, psychiatric symptoms are frequently associated with impaired cardiovascular function and perhaps also increased risk for cancer diseases. Pathophysiological basis of this comorbidity is not clearly understood. Molecular events involved, particularly factors modified by chronic stress exposure, may only be evaluated in animal models of depression.2. Present experiments were aimed to study parameters related to cardiovascular system (tyrosine hydroxylase (TH) gene expression in adrenal glands) and carcinogenesis (retinoic acid receptors in the liver) in the chronic mild stress model of depression.3. Chronic mild stress induced a rise in adrenal TH gene expression in both male and female rats. Gender dependent changes were found in retinoic acid receptor binding with stress-induced activation in females but not males. Ovariectomized animals exhibited higher retinoic acid receptor binding, slightly elevated TH mRNA levels and failed to respond to chronic mild stress exposure with further increase in TH mRNA levels. Similarly, chronic mild stress induced an anhedonic state manifested by decreased sucrose preference in control but not ovariectomized rats.4. Presented data document that central neurochemical and behavioral changes in animals exposed to chronic mild stress model of depression are associated with changes in adrenal TH gene expression and with gender dependent changes in retinoic acid receptor status in the liver. Such alterations may participate in the development of pathological changes and could participate on increased risk for cardiovascular and oncologic comorbidity in depressive patients.     

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH-SY5Y Human Neuroblastoma Cells

Abstract: Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca²⁺ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca²⁺ response to 1 m M carbachol is ∼50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.