Hirsutism, virilism, polycystic ovarian disease, and the steroid-gonadotropin-feedback system: a career retrospective.

This career retrospective describes how the initial work on the mechanism of hormone action provided the tools for the study of hirsutism, virilism, and polycystic ovarian disease. After excessive ovarian and or adrenal androgen secretion in polycystic ovarian disease had been established, the question whether the disease was genetic or acquired, methods to manage hirsutism and methods for the induction of ovulation were addressed. Recognizing that steroid gonadotropin feedback was an important regulatory factor, initial studies were done on the secretion of LH and FSH in the ovulatory cycle. This was followed by the study of basic mechanisms of steroid-gonadotropin feedback system, using castration and steroid replacement and the events surrounding the natural onset of puberty. Studies in ovariectomized rats showed that progesterone was a pivotal enhancer of estrogen-induced gonadotropin release, thus accounting for the preovulatory gonadotropin surge. The effects of progesterone were manifested by depletion of the occupied estrogen receptors of the anterior pituitary, release of hypothalamic LHRH, and inhibition of enzymes that degrade LHRH. Progesterone also promoted the synthesis of FSH in the pituitary. The 3α,5α-reduced metabolite of progesterone brought about selective LH release and acted using the GABA(A) receptor system. The 5α-reduced metabolite of progesterone brought about selective FSH release; the ability of progesterone to bring about FSH release was dependent on its 5α-reduction. The GnRH neuron does not have steroid receptors; the steroid effect was shown to be mediated through the excitatory amino acid glutamate, which in turn stimulated nitric oxide. These observations led to the replacement of the long-accepted belief that ovarian steroids acted directly on the GnRH neuron by the novel concept that the steroid feedback effect was exerted at the glutamatergic neuron, which in turn regulated the GnRH neuron. The neuroprotective effects of estrogens on brain neurons are of considerable interest.

Enhanced glutamatergic and decreased GABAergic synaptic appositions to GnRH neurons on proestrus in the rat: modulatory effect of aging.

BACKGROUND: Previous work by our lab and others has implicated glutamate as a major excitatory signal to gonadotropin hormone releasing hormone (GnRH) neurons, with gamma amino butyric acid (GABA) serving as a potential major inhibitory signal. However, it is unknown whether GABAergic and/or glutamatergic synaptic appositions to GnRH neurons changes on the day of the proestrous LH surge or is affected by aging. METHODOLOGY/PRINCIPAL FINDINGS: To examine this question, synaptic terminal appositions on GnRH neurons for VGAT (vesicular GABA transporter) and VGLUT2 (vesicular glutamate transporter-2), markers of GABAergic and glutamatergic synaptic terminals, respectively, was examined by immunohistochemistry and confocal microscopic analysis in young and middle-aged diestrous and proestrous rats. The results show that in young proestrous rats at the time of LH surge, we observed reciprocal changes in the VGAT and VGLUT2 positive terminals apposing GnRH neurons, where VGAT terminal appositions were decreased and VGLUT2 terminal appositions were significantly increased, as compared to young diestrus control animals. Interestingly, in middle-aged cycling animals this divergent modulation of VGAT and VGLUT2 terminal apposition was greatly impaired, as no significant differences were observed between VGAT and VGLUT2 terminals apposing GnRH neurons at proestrous. However, the density of VGAT and VGLUT2 terminals apposing GnRH neurons were both significantly increased in the middle-aged animals. CONCLUSIONS/SIGNIFICANCE: In conclusion, there is an increase in glutamatergic and decrease in GABAergic synaptic terminal appositions on GnRH neurons on proestrus in young animals, which may serve to facilitate activation of GnRH neurons. In contrast, middle-aged diestrous and proestrous animals show a significant increase in both VGAT and VGLUT synaptic terminal appositions on GnRH neurons as compared to young animals, and the cycle-related change in these appositions between diestrus and proestrus that is observed in young animals is lost.

A molecular guidance system based upon target genes, nuclear receptors and ligands applied to drug discovery and prediction of toxicity.

A molecular guidance system useful in drug design is described in which nuclear receptors position ligands in intercalation sites in responsive genes. Evidence is based upon positions of agonists in receptors and the transcriptional activity of a designed estrogen that is 3 times more potent than the steroid hormone estradiol.

PELP1--a novel estrogen receptor-interacting protein.

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) is a novel estrogen receptor (ER)-interacting protein that has been implicated to be important for mediation of both the genomic and nongenomic signaling of 17beta-estradiol (E2). PELP1 contains ten nuclear receptor-interacting boxes (LXXLL motifs), which allow it to interact with ER and other nuclear hormone receptors, a zinc finger, a glutamic acid-rich domain, and two proline-rich domains. The proline-rich regions contain several consensus PXXP motifs, which allow PELP1 to couple the ER with SH3 domain-containing kinase signaling proteins, such as Src and PI3K P85 regulatory subunit. PELP1 is expressed in many different brain regions, including the hippocampus, hypothalamus, and cerebral cortex. Further work has demonstrated that PELP1 is colocalized with ER-alpha in neurons in various brain regions. PELP1 is primarily expressed in neurons, with some expression also observed in glia. Subcellular localization studies revealed that PELP1 is highly localized in the cell nucleus of neurons, with some cytoplasm localization as well, and PELP1 is also localized at synaptic sites. Work in other tissues has demonstrated that PELP1 is critical for nongenomic and genomic signaling by E2, as PELP1 knockdown studies significantly attenuates E2-induced activation of ERK and Akt signaling pathways, and inhibits E2 genomic transcriptional effects on gene expression in breast cancer cells. Preliminary studies in the brain, suggests that similar roles may exist for PELP1 in the brain, but this remains to be established, and further work to characterize the precise roles and functions of PELP1 in the brain are needed.

Rapid estrogen signaling in the brain.

Estrogen has multiple actions in the brain to modulate homeostasis, synaptic plasticity/cognition and neuroprotection. While many of these actions undoubtedly involve mediation via the classical genomic mechanism of regulation of transcription of genes via estrogen nuclear receptors, there has been growing interest in the rapid nongenomic effects of estrogen and the role they may play in the neural actions of estrogen. In this review, we will focus on these rapid nongenomic actions of estrogen in the brain and discuss the potential physiological significance of these actions. The evidence for rapid estrogen regulation of cell signaling pathways, including calcium, ion channel and kinase signaling pathways in the brain will be reviewed, as will evidence derived from plasma-membrane impermeable estrogen-peptide conjugates in the regulation of these cell signaling pathways. Evidence supporting classical and nonclassical estrogen receptor localization to the plasma membrane of neurons will also be reviewed, including the putative new membrane estrogen G-protein-coupled receptor, GPR30. Precisely how membrane estrogen receptors couple to kinase signaling pathways is unclear, but we will discuss the latest findings on estrogen receptor-interacting scaffold proteins, such as MNAR/PELP1, striatin and p130Cas, which are capable of linking estrogen receptors and kinases such as Src and PI3K, to potentially mediate estrogen-induced kinase signaling. Finally, we will review the growing evidence that rapid membrane-mediated effects of estrogen play an important physiological role in the neural actions of estrogen in the brain, including estrogen feedback control and modulation of homeostasis, regulation of synaptic plasticity/cognition, and estrogen-mediated neuroprotection.

Tamoxifen neuroprotection in cerebral ischemia involves attenuation of kinase activation and superoxide production and potentiation of mitochondrial superoxide dismutase.

The purpose of this study was to enhance our understanding of the mechanisms of neuronal death after focal cerebral ischemia and the neuroprotective effects of tamoxifen (TMX). The phosphorylation state of 31 protein kinases/signaling proteins and superoxide anion (O(2)(-)) production in the contralateral and ipsilateral cortex was measured after permanent middle cerebral artery occlusion (pMCAO) in ovariectomized rats treated with placebo or TMX. The study revealed that pMCAO modulated the phosphorylation of a number of kinases/proteins in the penumbra at 2 h after pMCAO. Of significant interest, phospho-ERK1/2 (pERK1/2) was elevated significantly after pMCAO. TMX attenuated the elevation of pERK1/2, an effect correlated with reduced infarct size. In situ detection of O(2)(-) production showed a significant elevation at 1-2 h after pMCAO in the ischemic cortex with enhanced oxidative damage detected at 24 h. ERK activation may be downstream of free radicals, a suggestion supported by the findings that cells positive for O(2)(-) had high pERK activation and that a superoxide dismutase (SOD) mimetic, tempol, significantly attenuated pERK activation after MCAO. TMX treatment significantly reduced the MCAO-induced elevation of O(2)(-) production, oxidative damage, and proapoptotic caspase-3 activation. Additionally, pMCAO induced a significant reduction in the levels of manganese SOD (MnSOD), which scavenge O(2)(-), an effect largely prevented by TMX treatment, thus providing a potential mechanistic basis for the antioxidant effects of TMX. As a whole, these studies suggest that TMX neuroprotection may be achieved via an antioxidant mechanism that involves enhancement of primarily MnSOD levels, with a corresponding reduction of O(2)(-) production, and downstream kinase and caspase-3 activation.

Curcumin suppresses growth and chemoresistance of human glioblastoma cells via AP-1 and NFkappaB transcription factors.

Malignant gliomas are a debilitating class of brain tumors that are resistant to radiation and chemotherapeutic drugs, contributing to the poor prognosis associated with these tumors. Over-expression of transcription factors such as NFkappaB and AP-1 contribute to the enhanced glioma survival, radioresistance, and chemoresistance. Curcumin, which may inhibit these pathways, was therefore investigated for a potential therapeutic role in glioma. The effect of curcumin on glioma survival was investigated in human (T98G, U87MG, and T67) and rat (C6) glioma cell lines. The ability of curcumin to overcome glioma cell radioresistance and chemoresistance was also explored. Curcumin reduced cell survival in a p53- and caspase-independent manner, an effect correlated with the inhibition of AP-1 and NFkappaB signaling pathways via prevention of constitutive JNK and Akt activation. Curcumin-sensitized glioma cells to several clinically utilized chemotherapeutic agents (cisplatin, etoposide, camptothecin, and doxorubicin) and radiation, effects correlated with reduced expression of bcl-2 and IAP family members as well as DNA repair enzymes (MGMT, DNA-PK, Ku70, Ku80, and ERCC-1). These findings support a role for curcumin as an adjunct to traditional chemotherapy and radiation in the treatment of brain cancer.

Small molecule intercalation with double stranded DNA: implications for normal gene regulation and for predicting the biological efficacy and genotoxicity of drugs and other chemicals.

The binding of small molecules to double stranded DNA including intercalation between base pairs has been a topic of research for over 40 years. For the most part, however, intercalation has been of marginal interest given the prevailing notion that binding of small molecules to protein receptors is largely responsible for governing biological function. This picture is now changing with the discovery of nuclear enzymes, e.g. topoisomerases that modulate intercalation of various compounds including certain antitumor drugs and genotoxins. While intercalators are classically flat, aromatic structures that can easily insert between base pairs, our laboratories reported in 1977 that a number of biologically active compounds with greater molecular thickness, e.g. steroid hormones, could fit stereospecifically between base pairs. The hypothesis was advanced that intercalation was a salient feature of the action of gene regulatory molecules. Two parallel lines of research were pursued: (1) development of technology to employ intercalation in the design of safe and effective chemicals, e.g. pharmaceuticals, nutraceuticals, agricultural chemicals; (2) exploration of intercalation in the mode of action of nuclear receptor proteins. Computer modeling demonstrated that degree of fit of certain small molecules into DNA intercalation sites correlated with degree of biological activity but not with strength of receptor binding. These findings led to computational tools including pharmacophores and search engines to design new drug candidates by predicting desirable and undesirable activities. The specific sequences in DNA into which ligands best intercalated were later found in the consensus sequences of genes activated by nuclear receptors implying intercalation was central to their mode of action. Recently, the orientation of ligands bound to nuclear receptors was found to match closely the spatial locations of ligands derived from intercalation into unwound gene sequences suggesting that nuclear receptors may be guiding ligands to DNA with remarkable precision. Based upon multiple lines of experimental evidence, we suggest that intercalation in double stranded DNA is a ubiquitous, natural process and a salient feature of the regulation of genes. If double stranded DNA is proven to be the ultimate target of genomic drug action, intercalation will emerge as a cornerstone of the future discovery of safe and effective pharmaceuticals.

Induction of transforming growth factor-beta1 by basic fibroblast growth factor in rat C6 glioma cells and astrocytes is mediated by MEK/ERK signaling and AP-1 activation.

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) play an important role in proliferation, differentiation, and survival of malignant gliomas and in normal glial cell biology. Because of these critical roles, potential interactions between these key growth factors were investigated. We previously demonstrated that bFGF potently stimulates TGF-beta1 release from rat glioma cells. The purpose of the present study was to elucidate the mechanism(s) of this regulatory effect, establish its functional importance, and examine whether it extends to nontransformed rat hypothalamic astrocytes (RHA). The results revealed that RHA express the high-affinity FGF(1-4) receptors, and similarly to glioma cells, bFGF stimulated TGF-beta1 release in an isoform-specific manner. A mediatory role for ERK signaling in bFGF-induced TGF-beta release was suggested by the fact that MEK1 inhibition prevented this effect. Additionally, bFGF enhanced MEK1/2 phosphorylation and ERK activation/nuclear translocation, which culminated in increased activity of AP-1-mediated gene transcription. bFGF markedly induced TGF-beta1 mRNA levels in an isoform-specific manner, an effect that was dependent on MEK/ERK/AP-1 signaling. Functionally, bFGF-induced proliferation of glioma cells was attenuated by MEK/ERK inhibition or immunoneutralization of TGF-beta1, suggesting that this pathway may have important implications for brain tumor progression.

Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications.

Estrogen is an important hormone signal that regulates multiple tissues and functions in the body. This review focuses on the neurotrophic and neuroprotective actions of estrogen in the brain, with particular emphasis on estrogen actions in the hippocampus, cerebral cortex and striatum. Sex differences in the risk, onset and severity of neurodegenerative disease such as Alzheimer's disease, Parkinson's disease and stroke are well known, and the potential role of estrogen as a neuroprotective factor is discussed in this context. The review assimilates a complex literature that spans research in humans, non-human primates and rodent animal models and attempts to contrast and compare the findings across species where possible. Current controversies regarding the Women's Health Initiative (WHI) study, its ramifications, concerns and the new studies needed to address these concerns are also addressed. Signaling mechanisms underlying estrogen-induced neuroprotection and synaptic plasticity are reviewed, including the important concepts of genomic versus nongenomic mechanisms, types of estrogen receptor involved and their subcellular targeting, and implicated downstream signaling pathways and mediators. Finally, a multicellular mode of estrogen action in the regulation of neuronal survival and neurotrophism is discussed, as are potential future directions for the field.

Cloning, distribution, and colocalization of MNAR/PELP1 with glucocorticoid receptors in primate and nonprimate brain.

MNAR/PELP1 (see text) is a newly identified scaffold protein/coactivator initially thought to modulate nongenomic and genomic actions of the estrogen receptor; however, it has been recently shown to interact with multiple steroid receptors, including androgen and glucocorticoid receptors. In the present study, we cloned the monkey MNAR/PELP1 gene, deduced its domain structure, examined its localization pattern and colocalization with glucocorticoid receptor in monkey brain, and determined its subcellular localization. PCR-based cloning of MNAR/PELP1 from monkey brain produced a transcript of approximately 3.4 kb which showed high homology to the human and rat MNAR/PELP1 gene. Domain analysis showed that all the key steroid-receptor-interacting (LXXLL) domains, SH3-interacting (PXXP) domains and several C-terminal glutamic-acid-rich clusters, as well as various kinase domains are conserved in the monkey MNAR/PELP1 gene. Anatomical mapping of MNAR/PELP1 immunoreactivity in several regions of the monkey brain showed a similar pattern of MNAR/PELP1 distribution as previously observed in rat and mouse brains. MNAR/PELP1 also showed an absolute colocalization with glucocorticoid receptors in both primate and nonprimate brain, including those regions of the brain, where other steroid receptors are not significantly expressed, such as hippocampus, striatum, and thalamus - suggesting that MNAR/PELP1 may modulate glucocorticoid actions in the brain. Finally, ultrastructural electron microscopic studies showed that MNAR/PELP1-reactive gold particles are located within nucleus, cytoplasm, dendritic/synaptic terminals, and astrocytic processes. As a whole, the studies demonstrate that MNAR/PELP1 is expressed and colocalizes with glucocorticoid receptors in monkey and rat brains and may have multiple cellular functions based on its subcellular localizations.

Role of astrocytes in reproduction and neuroprotection.

Hypothalamic astrocytes secrete TGF-beta and 3 alpha,5 alpha-tetrahydro progesterone (3 alpha,5 alpha-THP) in culture. When the astrocyte-conditioned medium (ACM) was incubated with the hypothalamic cell line GT1-7, it resulted in the secretion of GnRH. Immunoneutralization with TGF-beta antibody or ultra-filteration with a 10 kDa cut off filter resulted in attenuation of the GnRH releasing ability of ACM, indicating that TGF-beta was a major factor involved with GnRH release. Treatment with estrogens increases TGF-beta secretion. These observations indicate a significant role of astrocytes in GnRH secretion. Serum-deprivation results in the death of GT1-7 neurons in culture and addition of ACM or TGF-beta to the culture, attenuates cell death. The mechanism of protection from cell death appears to involve phosphorylation of MKK4, JNK, c-Jun(Ser63), and enhancement of AP-1 binding. Co-administration of JNK inhibitors, but not MEK inhibitors attenuated ACM or TGF-beta-induced c-Jun(Ser63) phosphorylation and their neuroprotective effects. These studies suggest that astrocytes can protect neurons, at least in part, by the release of TGF-beta and activation of a c-Jun/AP-1 protective pathway.

Cloning, expression, and localization of MNAR/PELP1 in rodent brain: colocalization in estrogen receptor-alpha- but not in gonadotropin-releasing hormone-positive neurons.

MNAR/PELP1 is a recently identified scaffold protein in the human that modulates the nongenomic activity of estrogen receptors by facilitating linkage/cross talk with the Src/Erk activation cascade. We report herein the cloning of rat MNAR/PELP1 and provide new information concerning its distribution in the female rat brain and its degree of colocalization with estrogen receptor-alpha (ER-alpha) and GnRH. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence and retained all the key binding motifs (PXXP, LXXLL, and glutamic acid clusters) in its primary structure that are known to be critical for its interaction with Src and steroid receptors. RT-PCR revealed that the MNAR/PELP1 transcript is expressed in many regions of the brain, and immunohistochemistry studies showed intense MNAR/PELP1 immunoreactivity (MNAR/PELP1-ir) in areas such as the hypothalamus, cerebral cortex, hippocampus, amygdala, and cerebellum. MNAR/PELP1-ir principally localized in the nucleus, but some cytoplasmic and plasma membrane-associated staining was also observed. MNAR/PELP1-ir was also primarily neuronal, although some localization in glia cells was observed in select brain regions. Colocalization studies revealed that a majority of ER-alpha-positive cells in the brain colocalized MNAR/PELP1-ir. In contrast, MNAR/PELP1-ir rarely colocalized in GnRH neurons. In conclusion, the current study provides evidence that MNAR/PELP1 is expressed in key neural tissues of the rat brain that are known targets of steroid action, that its expression is primarily neuronal, and that MNAR/PELP1-ir is strongly colocalized in ER-alpha, but not GnRH neurons in the rodent brain.

Neuroprotection by stem cell factor in rat cortical neurons involves AKT and NFkappaB.

Stem cell factor (SCF) is a highly expressed cytokine in the central nervous system. In the present study, we demonstrate a neuroprotective role for SCF and its tyrosine kinase receptor, c-kit, against camptothecin-induced apoptosis and glutamate excitotoxicity in rat cortical neurons. This protection was blocked by pharmacological or molecular inhibition of either the MEK/ERK or PI3K/Akt signaling pathways. The importance of these pathways was further confirmed by the activation of both ERK, in a MEK-dependent manner, and Akt, via PI3K. Activation of Akt increased the binding of the p50 and p65 subunits of NFkappaB, which was also important for neuroprotection. Akt inhibition prevented NFkappaB binding, suggesting a role for Akt in SCF-induced NFkappaB. Pharmacological inhibition of NFkappaB or dominant negative IkappaB also prevented neuroprotection by SCF. SCF up-regulated the anti-apoptotic genes, bcl-2 and bcl-xL in an NFkappaB-dependent manner. Together, these findings demonstrate a neuroprotective role for SCF in cortical neurons, an effect that was mediated by Akt and ERK, as well as NFkappaB-mediated gene transcription. SCF represents a novel therapeutic target in the treatment of neurodegenerative disease.

The aging reproductive neuroendocrine axis.

It is well known that the reproductive system is one of the first biological systems to show age-related decline. While depletion of ovarian follicles clearly relates to the end of reproductive function in females, evidence is accumulating that a hypothalamic defect is critical in the transition from cyclicity to acyclicity. This minireview attempts to present a concise review on aging of the female reproductive neuroendocrine axis and provide thought-provoking analysis and insights into potential future directions for this field. Evidence will be reviewed, which shows that a defect in pulsatile and surge gonadotropin hormone-releasing hormone (GnRH) secretion exists in normal cycling middle-aged female rats, which is thought to explain the significantly attenuated pulsatile and surge luteinizing hormone (LH) secretion at middle-age. Evidence is also presented, which supports the age-related defect in GnRH secretion as being due to a reduced activation of GnRH neurons. Along these lines, stimulation of GnRH secretion by the major excitatory transmitter glutamate is shown to be significantly attenuated in middle-aged proestrous rats. Corresponding age-related defects in other major excitatory regulatory factors, such as catecholamines, neuropeptide Y, and astrocytes, have also been demonstrated. Age-related changes in hypothalamic concentrations of neurotransmitter receptors, steroid receptors, and circulating steroid hormone levels are also reviewed, and discussion is presented on the complex interrelationships of the hypothalamus-pituitary-ovarian (HPO) axis during aging, with attention to how a defect in one level of the axis can induce defects in other levels, and thereby potentiate the dysfunction of the entire HPO axis.

Expression of glutamate receptor subunits in the hypothalamus of the female rat during the afternoon of the proestrous luteinizing hormone surge and effects of antiprogestin treatment and aging.

The excitatory transmitter, glutamate has been implicated in the control of reproduction, hormone secretion and neuroendocrine regulation. The present study examined whether the hypothalamic expression of three key ionotropic glutamate receptor subunits (NMDAR1, GluR1 and GluR6) fluctuates significantly on proestrus in the rat, and whether treatment with the antiprogestin, RU486 affected glutamate receptor subunit expression. The studies revealed that NMDAR1, GluR1 and GluR6 mRNA levels in the mediobasal hypothalamus (MBH) and preoptic area (POA) fluctuate little throughout the day of proestrus. However, treatment with the antiprogestin, RU486 induced a significant elevation of GluR6 mRNA levels at 14.00 and 16.00 h on proestrus in the MBH, suggesting that endogenous progesterone (P4) may act to inhibit hypothalamic GluR6 levels. In support of this suggestion, exogenous P4 treatment to estrogen (E2)-primed ovariectomized (ovx) rats significantly suppressed GluR6 mRNA levels in the afternoon (12.00-16.00 h) in the MBH, and at 12.00 h in the POA, which preceded LH surge induction. Likewise, temporal examination of hypothalamic GluR6 protein levels in E2 + P4-treated young and middle-aged ovx rats revealed an early elevation from 12.00 to 14.00 h, which was followed by a fall from 16.00 to 20.00 h. The early elevation of GluR6 protein levels was most pronounced in the POA of the young rat, and this elevation was markedly attenuated in the middle-aged rat. As a whole, the studies suggest that glutamate receptor expression fluctuates little on proestrus in the hypothalamus, but that expression of the kainate GluR6 receptor subunit may be modulated by progesterone and aging.

Differential expression of the peripheral benzodiazepine receptor and gremlin during adipogenesis.

This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3-L1. 3T3-L1 cells were treated with dexamethasone, isobutyl-1-methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time-points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day 10 (d10), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down-regulated and 10 up-regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up-regulated on d3 and d10 were identified by sequence analysis to be lipoprotein lipase, a well-known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d10, was identified as DRM/gremlin, a bone morphogenetic protein antagonist. Finally, a sixth candidate that was increased at d3 and d10 was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.