Mechanisms Of Hormonal Regulation Research Articles

Mechanisms of Hormonal Regulation of the Sodium Chloride Cotransporter

The thiazide‐sensitive sodium chloride cotransporter (NCC) is one of the key determinants of salt balance and thus systemic blood pressure. Aldosterone and Angiotensin II appear to regulate NCC. However, in contrast to other important effectors of salt balance we know little about how hormonal signals alter activity of this cotransporter. Data demonstrates the role of the ERK1/2 MAPK (Extra‐cellular signal‐Regulated Kinases 1 and 2 Mitogen‐Activated Protein Kinases) pathway in the ubiquitination and endocytosis of NCC. With‐No‐Lysine Kinase 1 and 4 (WNK1 and WNK4), Ste20‐related proline alanine‐rich kinase (SPAK) and Oxidative Stress Response‐1 (OSR1) are also important regulators of NCC, and recent publications suggests that they may be linked to hormonal effects on NCC. However, the examination of links between hormones and the intracellular pathways that mediate the effects of hormones on NCC is only now beginning. Our recent data provides evidence that NCC is subject to hormonal regulation that proceeds through modulation of kinases.

Lactogenic Hormonal Induction of Long Distance Interactions between β-Casein Gene Regulatory Elements

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

The equine placenta and equine chorionic gonadotrophin--an overview.

Chorionic gonadotrophins seem to be unique for primate and equid species. Unlike primates, the equine conceptus does not implant in the maternal uterine endometrium until around day 37 of pregnancy. At this time specialized cells of the trophoblast, organized in the embryonic girdle, invade the endometrium and become established in the endometrial stroma, forming the so-called endometrial cups. This migration of girdle cells is accompanied by their morphological transformation into large decidual-like cells and by the appearance of a gonadotrophic hormone in the mare's blood. There is convincing evidence today that the hormone is of chorionic origin; therefore the term equine Chorionic Gonadotrophin (eCG) seems to be more appropriate than the formerly used term Pregnant Mare Serum Gonadotrophin (PMSG). Secretion of eCG peaks between days 60 and 80 in pregnant mares, to decline gradually until day 130 of gestation, with pronounced inter-individual variation. There appear to be no hormonal regulatory mechanisms controlling eCG synthesis and secretion, suggesting that the size and the morphology of the endometrial cups are the limiting factors. Equine CG is a glycoprotein hormone, composed of noncovalently bound alpha- and beta-subunits. The alpha-subunit consists of 96 amino acids and is identical for eCG and the pituitary hormones eLH, eFSH, and eTSH. The beta-subunit is similar to beta-hCG in that both have a C-terminal extension. It is comprised of 149 amino acids and the peptide primary structure is identical to that of beta-eLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Detached barley leaves as a model for studying cytokinin control of plastid gene regulation

We suggest a design of experiment, which permits studying a differential regulation of chloroplast gene transcription by exogenous cytokinin, using detached first barley leaves. Such experiments are of great interest for investigation of molecular mechanisms of the chloroplast biogenesis hormonal regulation. It was demonstrated that a cytokinin control of plastid genome transcription depended on the plant age, a source of detached leaves, chloroplast position in the leaf blade, and the intensity and duration of leaf illumination. Transcription regulation was studied for the first barley leaves detached from 9-day-old barley plants grown in soil. The leaves were preincubated for 24 h in the light (270 μmol/(m2 s) and then transferred for 3 h on water or cytokinin solution (benzyladenine, 2.2 × 10−5 M) under similar lighting condition. Chloroplasts were isolated from the most sensitive to cytokinin apical leaf part and used for run-on transcription. From 26 tested genes, cytokinin activated the rate of transcription of 19 genes (by 2 to 9 times as compared with control) and did not affect transcription of 7 genes. Cytokinin-sensitive genes encode proteins of photosynthesis and the components of the plastid genome expression system. The results obtained allow transition to more detail studying the mechanisms of hormonal regulation of chloroplast genome transcription.

Systems Biology of Vascular Calcification

Vascular calcification, a prevalent and progressive disorder, involves numerous interactive, autocrine, paracrine, and endocrine regulatory mechanisms and is thus ideally suited for analysis using a systems approach. This approach focuses on creating quantitative, testable models of complex biological systems that take into consideration the time dimension. They are usually expressed as mathematical models, and because of their time dependence and complexity, they usually require computer simulation to determine predicted outcomes. Here, we provide an example of such a model used to analyze self-organization and mineralization of vascular stem cells, using partial differential equations capable of accurately predicting experimental outcomes. Such systems-based models are useful in many aspects of cardiovascular medicine.

Ovine fetal hormonal and hypothalamic neuroendocrine responses to maternal water deprivation at late gestation

Angiotensin II (Ang II), aldosterone, and arginine-vasopressin (AVP) are three major neuropeptides or hormones that are important in the control of body fluid regulation. Dehydration during pregnancy induces alterations in maternal–fetal fluid homeostasis. It is still not clear about effects and mechanisms of maternal water deprivation on fetal neuroendocine and hormonal responses. The present study deprived water from pregnant sheep at near-term for 24h and 48h, and determined maternal and fetal blood osmolality and sodium levels before and immediately after water deprivation. Fetal renal excretion and plasma hormones were measured. Fetal forebrain was analyzed for cellular activation marked with Fos and Fos-B. The results showed that maternal and fetal blood osmolality and sodium were increased by water deprivation. Maternal and fetal Ang II, aldosterone, and AVP levels were elevated by 24-h and 48-h water deprivation, while fetal plasma Ang I levels were increased only under the condition of 48-h water deprivation. Intensive Fos and Fos-B expression was detected in the median preoptic nuclei and paraventricular nuclei in the fetal brain following exposure to maternal water deprivation. Double labeling demonstrated that many Fos-positive cells were AVP-containing neurons in the fetal paraventricular nucleus. Together, the results suggest that neuroendocrine and hormonal regulatory mechanisms play a role in the control of body fluid homeostasis, and relatively matured and functional at the last third of gestation, as well as the fetal hypothalamus is functional in the control of the neuropeptide in response to maternal dehydration.

Структурна організація, експресія та регуляція експресії генів 6-фосфофрукто-2-кінази/фруктозо-2,6-бісфосфатази

A bifunctional PFKFB enzyme is a key regulator of glycolysis. Four different genes encode the synthesis of multiple PFKFB isoforms. We analyzed data concerning structural organization of PFKFB genes and alternative splice variants of PFKFB mRNA, as well as expression and molecular mechanisms of regulation of different PFKFB isoforms in malignant tumors and at hypoxia, and mechanisms of hormonal regulation. Hypoxia inducible factor (HIF)-dependent mechanisms of regulation of the expression of different PFKFB variants are presented and discussed. Keywords: PFKFB, expression regulation, mRNA, alternative splicing, HIF.

Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes.

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

Regulation of the epithelial sodium channel by membrane trafficking

The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle's syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in states as diverse as salt-sensitive hypertension, nephrosis, and pulmonary edema. ENaC activity in epithelial cells is highly regulated both by open probability and number of channels. Open probability is regulated by a number of factors, including proteolytic processing, while ENaC number is regulated by cellular trafficking. This review discusses current understanding of apical membrane delivery, cell surface stability, endocytosis, retrieval, and recycling of ENaC and the molecular partners that have so far been shown to participate in these processes. We review known sites and mechanisms of hormonal regulation of trafficking by aldosterone, vasopressin, and insulin. While many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.

Changes in Glial Fibrillary Acidic Protein (GFAP) Immonureactivity Reflect Neuronal States

The astroglial marker, glial fibrillary acidic protein (GFAP) was investigated by immunohistochemistry in various brain areas in order to see its fluctuations in various functional states. Different neuronal states were either experimentally induced or studied under physiological conditions. To produce experimental alterations the visual system was chosen as a model. Upon lesioning of the lateral geniculate body with the stereotaxic injection of ibotenic acid an increase in GFAP immunoreactivity could be induced in layers III and IV of the ipsilateral visual cortex where geniculo-cortical fibres terminate. Electron microscopy has revealed a synchronous degeneration of synaptic terminals and the hypertrophy of perisynaptic astrocyte processes. To study changes in the intact animal the effect of illumination was observed. In the lateral geniculate body the dorsal subnucleus was found immunonegative when studied at day and positive at night. Similar changes were observed in the suprachiasmatic nucleus. As to more generalized influences, the effect of gonadal steroids on the GFAP-reaction interpeduncular nucleus, an area not involved in hormonal regulatory mechanisms was studied. In males only castration could reduce constantly high GFAP immunoreactivity, whereas in females GFAP showed wide-range sexual cycle-related fluctuations. It was concluded that changes in GFAP immunoreactivity can indicate synaptic events within a circumscribed area of the brain.

Estradiol and Progesterone Regulate HIV Type 1 Replication in Peripheral Blood Cells

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.

Endocrine Disruptors and Abnormalities of Pubertal Development

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study.

Sexual dimorphism of body composition

Sexual dimorphism in human body composition is evident from fetal life, but emerges primarily during puberty. At birth, males have a similar fat mass to females but are longer and have greater lean mass. Such differences remain detectable during childhood; however, females enter puberty earlier and undergo a more rapid pubertal transition, whereas boys have a substantially longer growth period. After adjusting for dimorphism in size (height), adult males have greater total lean mass and mineral mass, and a lower fat mass than females. These whole-body differences are complemented by major differences in tissue distribution. Adult males have greater arm muscle mass, larger and stronger bones, and reduced limb fat, but a similar degree of central abdominal fat. Females have a more peripheral distribution of fat in early adulthood; however, greater parity and the menopause both induce a more android fat distribution with increasing age. Sex differences in body composition are primarily attributable to the action of sex steroid hormones, which drive the dimorphisms during pubertal development. Oestrogen is important not only in body fat distribution but also in the female pattern of bone development that predisposes to a greater female risk of osteoporosis in old age. Disorders of sex development are associated with significant abnormalities of body composition, attributable largely to their impact on mechanisms of hormonal regulation.

Low-carbohydrate diets affect energy balance and fuel homeostasis differentially in lean and obese rats

In parallel with increased prevalence of overweight people in affluent societies are individuals trying to lose weight, often using low-carbohydrate diets. Nevertheless, long-term metabolic consequences of those diets, usually high in (saturated) fat, remain unclear. Therefore, we investigated long-term effects of high-fat diets with different carbohydrate/protein ratios on energy balance and fuel homeostasis in obese (fa/fa) Zucker and lean Wistar rats. Animals were fed high-carbohydrate (HC), high-fat (HsF), or low-carbohydrate, high-fat, high-protein (LC-HsF-HP) diets for 60 days. Both lines fed the LC-HsF-HP diet displayed reduced energy intake compared with those fed the HsF diet (Zucker, -3.7%) or the HC diet (Wistar rats, -12.4%). This was not associated with lower weight gain relative to HC fed rats, because of increased food efficiencies in each line fed HsF and particularly LC-HsF-HP food. Zucker rats were less glucose tolerant than Wistar rats. Lowest glucose tolerances were found in HsF and particularly in LC-HsF-HP-fed animals irrespective of line, but this paralleled reduced plasma adiponectin levels, elevated plasma resistin levels, higher retroperitoneal fat masses, and reduced insulin sensitivity (indexed by insulin-induced hypoglycemia) only in Wistar rats. In Zucker rats, however, improved insulin responses during glucose tolerance testing and tendency toward increased insulin sensitivities were observed with HsF or LC-HsF-HP feeding relative to HC feeding. Thus, despite adverse consequences of LC-HsF diets on blood glucose homeostasis, principal differences exist in the underlying hormonal regulatory mechanisms, which could have benefits for B-cell functioning and insulin action in the obese state but not in the lean state.

Novel mechanisms of growth hormone regulation: growth hormone-releasing peptides and ghrelin

Growth hormone secretion is classically modulated by two hypothalamic hormones, growth hormone-releasing hormone and somatostatin. A third pathway was proposed in the last decade, which involves the growth hormone secretagogues. Ghrelin is a novel acylated peptide which is produced mainly by the stomach. It is also synthesized in the hypothalamus and is present in several other tissues. This endogenous growth hormone secretagogue was discovered by reverse pharmacology when a group of synthetic growth hormone-releasing compounds was initially produced, leading to the isolation of an orphan receptor and, finally, to its endogenous ligand. Ghrelin binds to an active receptor to increase growth hormone release and food intake. It is still not known how hypothalamic and circulating ghrelin is involved in the control of growth hormone release. Endogenous ghrelin might act to amplify the basic pattern of growth hormone secretion, optimizing somatotroph responsiveness to growth hormone-releasing hormone. It may activate multiple interdependent intracellular pathways at the somatotroph, involving protein kinase C, protein kinase A and extracellular calcium systems. However, since ghrelin has a greater ability to release growth hormone in vivo, its main site of action is the hypothalamus. In the current review we summarize the available data on the: a) discovery of this peptide, b) mechanisms of action of growth hormone secretagogues and ghrelin and possible physiological role on growth hormone modulation, and c) regulation of growth hormone release in man after intravenous administration of these peptides.

EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone

BackgroundProstate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells.ResultsThe studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells.ConclusionThis study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

Prevention, diagnosis and treatment of the Overtraining Syndrome

Successful training must involve overload but also must avoid the combination of excessive overload plus inadequate recovery. Athletes can experience short term performance decrement, without severe psychological, or lasting other negative symptoms. This Functional Overreaching (FOR) will eventually lead to an improvement in performance after recovery. When athletes do not sufficiently respect the balance between training and recovery, Non-Functional Overreaching (NFOR) can occur. The distinction between NFOR and the Overtraining Syndrome (OTS) is very difficult and will depend on the clinical outcome and exclusion diagnosis. The athlete will often show the same clinical, hormonal and other signs and symptoms. A keyword in the recognition of OTS might be ‘prolonged maladaptation’ not only of the athlete, but also of several biological, neurochemical, and hormonal regulation mechanisms. It is generally thought that symptoms of OTS, such as fatigue, performance decline, and mood disturbances, are more severe than those of NFOR. However, there is no scientific evidence to either confirm or refute this suggestion. One approach to understanding the aetiology of OTS involves the exclusion of organic diseases or infections and factors such as dietary caloric restriction (negative energy balance) and insufficient carbohydrate and/or protein intake, iron deficiency, magnesium deficiency, allergies, etc. together with identification of initiating events or triggers. In this paper we provide the recent status of possible markers for the detection of OTS. Currently several markers (hormones, performance tests, psychological tests, biochemical and immune markers) are used, but none of them meets all criteria to make its use generally accepted. We propose a “check list” that might help the physicians and sport scientists to decide on the diagnosis of OTS and to exclude other possible causes of underperformance.

Evolution of Sexual Dimorphism and Male Dimorphism in the Expression of Beetle Horns: Phylogenetic Evidence for Modularity, Evolutionary Lability, and Constraint

Beetle horns are enlarged outgrowths of the head or thorax that are used as weapons in contests over access to mates. Horn development is typically confined to males (sexual dimorphism) and often only to the largest males (male dimorphism). Both types of dimorphism result from endocrine threshold mechanisms that coordinate cell proliferation near the end of the larval period. Here, we map the presence/absence of each type of dimorphism onto a recent phylogeny for the genus Onthophagus (Coleoptera: Scarabaeidae) to explore how horn development has changed over time. Our results provide empirical support for several recent predictions regarding the evolutionary lability of developmental thresholds, including uncoupled evolution of alternative phenotypes and repeated fixation of phenotypes. We also report striking evidence of a possible developmental constraint. We show that male dimorphism and sexual dimorphism map together on the phylogeny; whenever small males have horns, females also have horns (and vice versa). We raise the possibility that correlated evolution of these two phenomena results from a shared element in their endocrine regulatory mechanisms rather than a history of common selection pressures. These results illustrate the type of insight that can be gained only from the integration of developmental and evolutionary perspectives.

A T Lymphocyte-Specific Transcription Complex Containing RUNX1 Activates MHC Class I Expression

MHC class I expression is subject to both tissue-specific and hormonal regulatory mechanisms. Consequently, levels of expression vary widely among tissues, with the highest levels of class I occurring in the lymphoid compartment, in T cells and B cells. Although the high class I expression in B cells is known to involve the B cell enhanceosome, the molecular basis for high constitutive class I expression in T cells has not been explored. T cell-specific genes, such as TCR genes, are regulated by a T cell enhanceosome consisting of RUNX1, CBFbeta, LEF1, and Aly. In this report, we demonstrate that MHC class I gene expression is enhanced by the T cell enhanceosome and results from a direct interaction of the RUNX1-containing complex with the class I gene in vivo. T cell enhanceosome activation of class I transcription is synergistic with CIITA-mediated activation and targets response elements distinct from those targeted by CIITA. These findings provide a molecular basis for the high levels of MHC class I in T cells.

Hormonal regulation of lactate dehydrogenase-A through activation of protein kinase C pathways in MCF-7 breast cancer cells

Lactate dehydrogenase A (LDH-A) is hormonally regulated in rodents, and increased expression of LDH-A is observed during mammary gland tumorigenesis. The mechanisms of hormonal regulation of LDH-A were investigated using a series of deletion and mutant constructs derived from the rat LDH-A gene promoter. Results of these studies show that constructs containing the −92 to −37 region of the LDH-A promoter are important for basal and E2-induced transactivation, and mutation of the consensus CRE motif within this region results in significant loss of basal activity and hormone-responsiveness. Gel mobility shift assays using nuclear extracts from MCF-7 cells show that both CREB and ATF-1 interact with the CRE. Studies with kinase inhibitors show that E2-induced activation of this CRE is dependent on protein kinase C, and these data indicate that LDH-A is induced through a non-genomic pathway of estrogen action.