Alleviation of autophagy by knockdown of Beclin-1 enhances susceptibility of hippocampal neurons to proapoptotic signals induced by amino acid starvation.

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.

Differential topochemistry of three cationic amino acid transporter proteins, hCAT1, hCAT2 and hCAT3, in the adult human brain.

The cellular uptake of L-arginine and other cationic amino acids (such as L-lysine and L-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.

Tissue inhibitor of metalloproteinases II (TIMP-2) is an osteoanabolic factor in vitro and in vivo.

OBJECTIVE: Critical size defects (CSDs) of bone are defined as defects that do not heal spontaneously to new bone during the lifetime of an adult individual. In contrast, immature animals are capable to heal defects of identical size. It was our hypothesis that age-related paracrine effects are relevant for this difference in regeneration. METHODS: The pooled supernatant of primary rat calvarial osteoblast-like cell cultures (POBC) derived from prenatal or postnatal donors was concentrated and applied into CSDs of adult recipient organisms (n = 10). In addition, the supernatant of POBC derived from prenatal donors was pooled and purified by reverse-phase chromatography. Each pre-purified fraction was tested in a proliferation indicating bioassay. Peptide fractions containing proliferative activities were re-chromatographed and re-tested in a bioassay. Finally, a proliferative activity was purified, identified by sequence analysis and applied into CSDs of adult recipients. RESULTS: The application of POBC derived from prenatal donors resulted in osseous regeneration of a CSD in adult recipients, while the supernatant of postnatal donors had much smaller effects. The morphologic features resembled the spontaneous osseous healing of calvarial defects of the same size in immature organisms. The polypeptide "tissue inhibitor of metalloproteinases type II"(TIMP-2) was isolated from the supernatant of cultures of POBC derived from prenatal donors by measuring the induction of their proliferation. Additionally, the application of human TIMP-2 injected into calvarial CSDs of adult organisms resulted in osseous healing. CONCLUSION: We conclude that one component responsible for the healing effect of CSDs of POBC supernatants derived from prenatal donors is TIMP-2.

Transcription factor CREB and its stimulus-dependent phosphorylation in cell and explant cultures of the bovine subcommissural organ.

The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures.

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.

Direct comparison of the potency of three novel cAMP analogs to induce CREB-phophorylation in rat pinealocytes.

A modified analog of cyclic adenosine 3',5'-monophosphate (cAMP), Sp-adenosine-3',5'-monophosphorothioate, designed to be highly membrane-permeable and resistant towards phosphodiesterases was found to induce the phosphorylation of the cAMP-regulated transcription factor cyclic AMP-responsive element binding protein in cultured rat pinealocytes more efficiently than previously described cAMP analogs.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat.

BACKGROUND: Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS: RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS: GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION: GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Human natriuretic peptides exhibit antimicrobial activity.

Here we describe a novel function for members of the well-characterized human natriuretic peptide family. Human "brain-type natriuretic peptide" (hBNP-32) as well as other members of this peptide class are antimicrobially active against Gram-positive and Gram-negative bacteria and yeast in a dose-dependent manner. This activity of natriuretic peptides is comparable to that of known antimicrobial peptides such as casocidins or magainins.

The human PER1 gene is transcriptionally regulated by multiple signaling pathways.

The mammalian period (Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the human PER1 (hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenous hPER1 expression and hPER1 promoter-driven reporter gene activity in a dose-dependent manner. Our results suggest that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation of hPER1 appears to be significant for rapid adaptation to changing environmental conditions.

Cyclic nucleotide analogs as biochemical tools and prospective drugs.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealth of synthetic analogs of cAMP and cGMP, only a few have been explored with regard to their therapeutic potential. Some of the first-generation cyclic nucleotide analogs were promising enough to be tested as drugs, for instance N(6),O(2)'-dibutyryl-cAMP and 8-chloro-cAMP (currently in clinical Phase II trials as an anticancer agent). Moreover, 8-bromo and dibutyryl analogs of cAMP and cGMP have become standard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers of adenosine 3',5'-cyclic monophosphorothioate and guanosine 3',5'-cyclic monophosphorothioate as competitive inhibitors of cAMP- and cGMP-dependent protein kinases, as well as subsequent development of related analogs, has proven very useful for studying the molecular basis of signal transduction. These analogs exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity. Furthermore, better understanding of signaling pathways and ligand/protein interactions has led to new therapeutic strategies. For instance, Rp-8-bromo-adenosine 3',5'-cyclic monophosphorothioate is employed against diseases of the immune system. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research, but also highlights some historical findings in the field.

Antisense experiments reveal molecular details on mechanisms of ICER suppressing cAMP-inducible genes in rat pinealocytes.

In the rat pineal gland neuronal signals determine the rhythmic synthesis of the hormone melatonin. Norepinephrine (NE) is the principal neurotransmitter that drives hormone synthesis by activating the cAMP signaling pathway. This activation depends on transcriptional and posttranscriptional regulatory mechanisms. The cAMP-dependent transcriptional regulation of the rate-limiting enzyme of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) involves the activating transcription factor (TF) CREB and the inhibitory TF ICER. By silencing elements of this cAMP-dependent neuroendocrine transduction cascade we wished to gain further insight into the role of ICER in the regulation of gene expression in rat pineal gland. Inhibition of specific kinases in primary pinealocyte cultures showed that ICER induction depends pivotally on the activation of cAMP-dependent protein kinase II. Eliminating ICER's impact by transfecting antisense constructs into pinealocytes revealed a predominant beta-adrenergic mechanism in regulating a cotransfected CRE-inducible reporter gene and notably, also the endogenous AA-NAT gene. Deciphering molecular details of the cAMP-dependent gene expression in mammalian pinealocytes provides a basis for understanding the general architecture of this signaling pathway that serves adaptive processes ubiquitously in the organism.

Neurofilament H immunoreaction in oligodendrogliomas as demonstrated by a new polyclonal antibody.

We have characterized a new polyclonal antibody against heavy chain (H) of neurofilament which can be used to demonstrate neurofilament H in normal brain tissue and oligodendroglioma cells immunocytochemically and immunochemically. Using this antibody we found neurofilament H-immunoreactive tumor cells in 13 oligodendrogliomas (6 WHO grade II, 7 WHO grade III) out of 84 oligodendrogliomas investigated (59 WHO grade II and 25 WHO grade III). Double immunolabeling and confocal laser scanning microscopy showed colocalization of neurofilament H and glial fibrillary acidic protein in certain oligodendroglioma cells. Colocalization of neurofilament and synaptophysin was observed only rarely. The results support the notion that oligodendrogliomas consists of a heterogeneous cell population displaying various stages of differentiation and dedifferentiation. The occurrence of neurofilament H-immunoreactive tumor cells in oligodendrogliomas is not related to the survival of the patients.

Analyses of signal transduction cascades reveal an essential role of calcium ions for regulation of melatonin biosynthesis in the light-sensitive pineal organ of the rainbow trout (Oncorhynchus mykiss).

Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.

Expression and cell-specific localization of the cholecystokinin B/gastrin receptor in the human stomach.

Gastrin stimulates gastric acid secretion by acting on the cholecystokinin B/gastrin receptor (CCK-BR). The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in man by immunohistochemistry. The aim of the present study is to characterize the precise cellular location of the CCK-BR in the human stomach. Polyclonal antisera were raised against different epitopes of the CCK-BR molecule and used for immunohistochemical investigations. CCK-BR mRNA was detected in paraffin tissue sections by the highly sensitive method of in situ reverse transcriptase-polymerase chain reaction (RT-PCR). Using immunohistochemistry, CCK-BR could successfully be localized in gastric parietal cells. In the majority of parietal cells, CCK-BR immunoreactivity was present a he basolateral cell membrane domain. In some parietal cells, a granular pattern of immunoreactivity was exclusively confined to the cytoplasm of the cells. CCK-BR mRNA was found in parietal cells and in enterochromaffin-like (ECL) cells by means of in situ RT-PCR. No expression of CCK-BR was found in the gastric antral mucosa. Our data support the concept that gastrin stimulates gastric acid secretion directly via CCK-B receptors on parietal cells and indirectly by inducing histamine release from histamine-containing ECL cells, which contributes to acid secretion by parietal cells.

CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

Inducible cyclic AMP early repressor protein in rat pinealocytes: a highly sensitive natural reporter for regulated gene transcription.

Rhythmic activity of arylalkylamine N-acetyltransferase (AANAT) determines melatonin synthesis in rat pineal gland. The transcriptional regulation of AANAT involves the activating and inhibiting transcription factors of the cyclic AMP (cAMP)-signaling pathway, cAMP response element-binding protein and inducible cAMP early repressor (ICER), respectively. Activation of this pathway is centered around norepinephrine, stimulating beta(1)-adrenergic receptors, but various other transmitters can modulate melatonin biosynthesis. To compare the transcriptional impact of norepinephrine with that of other neurotransmitters on melatonin synthesis, we determined ICER protein levels in pinealocytes and, in parallel, hormone secretion. The dose-dependent inductions of ICER protein by norepinephrine, the beta(1)-adrenergic receptor agonist isoproterenol, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and adenosine are correlated to regulatory dynamics in melatonin production. Importantly, ICER protein induction required lower ligand concentrations than the induction of melatonin biosynthesis. Although neuropeptide Y, glutamate, and vasopressin altered norepinephrine-stimulated hormone production without affecting ICER levels, the activation of voltage-gated cation channels increased ICER without affecting hormone synthesis. Sensitivity and versatility of ICER induction in pinealocytes make these neuroendocrine cells a valuable model system in which to study molecular interactions determining a regulated gene expression.

Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis.

Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element-binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes the in vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis.

A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations.

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.

Signal transduction in the rodent pineal organ. From the membrane to the nucleus.

The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.

Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells.

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days Northern and Western blot analyses revealed that all regulatory subunit isoforms (RI alpha, RI beta, RII alpha, and RII beta) and catalytic subunits C alpha and C beta of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization but in decidualized ES cells, exposed to relaxin for more than 6 days a significant reduction of RI alpha protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as proliferating cell nuclear antigen is evenly expressed in undifferentiated and differentiated ES cell cultures. In transient transfections of undifferentiated ES cells, the dPRL promoter is activated by 8-bromo-cAMP and the C subunit (C beta) of PKA. This induction as well as the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells are effectively abolished by the coexpression of protein kinase inhibitor. We demonstrate that 332 bp of the dPRL promoter are sufficient to mediate full inducibility by cAMP. Activation of the dPRL promoter by cAMP in ES cells occurs in two steps: an initial weak induction within 12 h and a subsequent, much more pronounced induction after 12 h. The secondary induction is not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter and is absent from a uterine cell line that does not express the endogenous dPRL gene. The early response of the dPRL promoter depends upon a noncanonical CRE at position -12, as mutation of this sequence leads to abolition of the early, but not the delayed, induction. The major activation depends upon a different region within 332 bp of the dPRL promoter; is probably indirect, as it follows different kinetics compared to a classical CRE-mediated response; and is specific to ES cells.