Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel.

OBJECTIVE: Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel. MATERIALS AND METHODS: We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves. RESULTS: hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone. CONCLUSION: Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel.

Adiponectin attenuation of endocrine function within human term trophoblast cells.

The hormone adiponectin has been shown to be important in maintaining insulin sensitivity throughout the body, whereas potential effects on the placenta have not been assessed. Pregnancy constitutes a unique physiological environment in which metabolism has a profound effect on the health of both the mother and the developing fetus. It is imperative that a delicate balance in glucose delivery be maintained between maternal tissues and the fetal/placental unit. Adiponectin's role in regulating peripheral insulin responsiveness suggests it may be a factor in maintaining this balance during gestation as well. Examination of human cytotrophoblast cells revealed that mRNA for both adiponectin receptors, adipoR1 and adipoR2, are abundantly expressed at term. We were, however, unable to reliably detect mRNA for adiponectin in primary cytotrophoblasts. Expression of both receptors was maintained after induction of syncytium formation by exogenous epidermal growth factor treatment. Treatment of cytotrophoblasts with adiponectin resulted in a significant drop, as assessed by quantitative RT-PCR, in expression for a number of genes involved in the endocrine function of the placenta, including the chorionic gonadotropin subunits, placental lactogen, and some steroidogenic enzymes. Immunofluorescent staining for connexin 43 and desmoplakin in primary trophoblasts revealed that adiponectin does not inhibit syncytialization of trophoblast cells in culture. Taken together, these data describe a novel role for maternal adiponectin in regulating the placental environment. Determination of the effects of such adipokines on the maternal-fetal interface is increasingly important, because the incidence of pregnancies complicated by gestational diabetes remains a significant health problem in developed countries.

Developmental expression of the homeobox protein Distal-less 3 and its relationship to progesterone production in mouse placenta.

Distal-less 3 (Dlx3) is a homeobox factor that functions as a placental-specific transcriptional regulator. Dlx3 null mice (-/-) have compromised placental development and do not survive in utero past embryonic day (E) 9.5. The current studies were undertaken to examine the expression of Dlx3 in mouse placenta during gestation, and to determine whether Dlx3 was involved in placental progesterone production. Dlx3 was not detectable at E8.5 but was detected in E9.5 placenta with continuing but diminished expression through E15.5. Dlx3 immuno-localization was restricted to the labyrinth, was nuclear and was found in cytokeratin-positive cells. Previous studies in choriocarcinoma cell lines support the conclusion that Dlx3 is required for expression of 3'-hydroxysteroid dehydrogenase VI (3betaHSD VI), an obligate enzyme in the production of progesterone by trophoblast giant cells. In a rat trophoblast stem cell line (Rcho-1), Dlx3 expression was non-detectable in Rcho-1 cells induced to differ-entiate using mitogen withdrawal. In vitro progesterone production in placental cultures and 3betaHSD VI mRNA from Dlx3 (+/+), (+/-) and (-/-) mice were equivalent. In situ hybridization for 3betaHSD VI revealed mRNA expression restricted to trophoblast giants cells with no detectable expression in the labyrinth suggesting that Dlx3 and 3betaHSD VI were not colocalized within the placenta. These studies support the conclusion that Dlx3 protein expression is restricted to the labyrinth region of the murine placenta into late gestation and that Dlx3 does not appear to be expressed in trophoblast giant cells. Further, loss of Dlx3 was not correlated with synthesis of progesterone from E9.5 mouse placentas.

Component rotation and anterior knee pain after total knee arthroplasty.

All patients undergoing cruciate-retaining primary total knee arthroplasty for degenerative osteoarthritis at one center were studied prospectively. Clinical and radiographic followup was obtained at a minimum 5 years in 102 knees in 73 patients. Patients were asked specifically about the presence of the pain in the anterior aspect of the knee in the vicinity of the patella and rated the severity of the pain on a visual analog scale. Significant anterior knee pain rating at least 3 of 10 on the visual analog scale was present in 16 knees (13 patients). Eleven patients with 14 symptomatic knees agreed to undergo computed tomography scanning to accurately determine the rotation of the tibial and femoral components. The epicondylar axis and tibial tubercle were used as references using a previously validated technique. A control group of 11 asymptomatic patients (14 knees), matched for age, gender, and length of followup also underwent computed tomography scanning. All patients in both groups had normal axial alignment. There was a highly significant difference in tibial component rotation between the two groups with the patients with anterior knee pain averaging 6.2 degrees internal rotation compared with 0.4 degrees external rotation in the control group. There also was a significant difference in combined component rotation with the patients with anterior knee pain averaging 4.7 degrees internal rotation compared with 2.6 degrees external rotation in the control group. There was no significant difference in the degree of radiographic patellar tilt or patellar subluxation between the two groups. Patients with combined component internal rotation were more than five times as likely to experience anterior knee pain after total knee arthroplasty compared with those with combined component external rotation. Component malrotation is a significant factor in the development of anterior knee pain after total knee arthroplasty.

An early growth response protein (Egr) 1 cis-element is required for gonadotropin-releasing hormone-induced mitogen-activated protein kinase phosphatase 2 gene expression.

In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) activates all three major mitogen-activated protein kinase (MAPK) cascades. The MAPKs play key roles in transcriptional activation of GnRH-responsive genes. MAPK phosphatases (MKPs) are dual specificity protein phosphatases involved in feedback regulation of MAPK activity. Previous studies indicate that GnRH activates MKP-2 expression in gonadotropes, dependent upon activation of multiple MAPKs and discrete Ca(2+) signals. To further understand the transcriptional mechanism(s) of MKP-2 induction by GnRH, we studied the activity of a 198-nucleotide MKP-2 proximal promoter region that supports GnRH responsiveness in reporter gene assays. Functional analysis of the MKP-2 promoter confirmed a requirement for the protein kinase C-extracellular signal-regulated kinase (ERK) pathway and VGCC-derived Ca(2+) signals in transcriptional activation of the MKP-2 gene. However, the inhibitory effect of thapsigargin on MKP-2 protein expression previously identified was not mediated at the level of promoter activation, suggesting a distinct mechanism for the action of thapsigargin-sensitive Ca(2+) signals. MGRE (MKP-2 GnRH response element) within the MKP-2 promoter mediated promoter activation through the protein kinase C-ERK pathway. The zinc finger transcription factor Egr-1 was identified in the MGRE-binding complex. Egr-1/MGRE binding was induced by GnRH in an ERK-dependent manner. Transcriptional activity of Egr-1 protein was enhanced by GnRH treatment. In addition, overexpression of the Egr-interacting protein, NAB1, resulted in increased GnRH-stimulated MKP-2 gene transcription. Consistent with the putative role of Egr-1 in MKP-2 promoter regulation, Egr-1 protein expression closely correlated with the expression of MKP-2 protein in alpha T3-1 cells. Together, these data suggest that Egr-1 may be a key factor in mediating GnRH-dependent transcriptional activation of the MKP-2 gene.

Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

Patellar resurfacing in total knee arthroplasty. A prospective, randomized, double-blind study with five to seven years of follow-up.

BACKGROUND: Whether to resurface the patella during a primary total knee arthroplasty performed for the treatment of degenerative osteoarthritis remains a controversial issue. Parameters that have been suggested as being useful in guiding this decision include patient height and weight, the presence of anterior knee pain preoperatively, and the grade of chondromalacia encountered intraoperatively. The purpose of this study was to determine whether these parameters were predictive of the clinical result following total knee arthroplasty with or without patellar resurfacing. METHODS: Eighty-six patients (118 knees) undergoing primary total knee arthroplasty for the treatment of osteoarthritis were enrolled in a prospective, randomized, double-blind study. All patients received the same posterior-cruciate-sparing total knee prosthetic components. Patients were randomized to treatment with or without resurfacing of the patella. Evaluations consisted of the determination of a Knee Society clinical score, the completion of a patient satisfaction questionnaire, specific questions relating to patellofemoral symptoms, and radiographs. Sixty-seven patients (ninety-three knees) were followed for a minimum of five years (range, sixty to eighty-four months; average, 70.5 months). RESULTS: With the numbers available, there was no significant difference between the groups treated with and without resurfacing with regard to the overall Knee Society score or the pain and function subscores. Obesity, the degree of patellar chondromalacia, and the presence of preoperative anterior knee pain did not predict postoperative clinical scores or the presence of postoperative anterior knee pain. CONCLUSIONS: The occurrence of anterior knee pain could not be predicted with any clinical or radiographic parameter studied. On the basis of these results, it seems likely that postoperative anterior knee pain is related either to the component design or to the details of the surgical technique, such as component rotation, rather than to whether or not the patella is resurfaced.

Epidermal growth factor regulation of equine glycoprotein hormone alpha subunit expression in trophoblast cells.

Primates and equids are the only species known to express the placental glycoprotein hormone, chorionic gonadotropin (CG), a heterodimeric glycoprotein composed of an alpha subunit linked to a hormone-specific beta subunit. The regulatory mechanisms involved in the induction of equine glycoprotein alpha subunit gene expression have not been identified. Epidermal growth factor (EGF) receptor is known to transduce signals that alter a number of different cellular functions (cell proliferation, differentiation, hormone secretion, and gene regulation). In the present study, we investigated the regulation of the equine alpha subunit gene by EGF in trophoblasts. We found that 2800 base pairs of 5' flanking sequence from the equine alpha subunit promoter is sufficient for basal expression in human choriocarcinoma cells. Epidermal growth factor and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), increased transcriptional activity of the equine alpha subunit promoter (-2800/+21). These responses were blocked by pretreatment with bisindolylmaleimide-I, an inhibitor of PKC, suggesting an involvement of this pathway downstream of EGF. In addition, PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) pathway, completely blocked activation of the equine alpha promoter by PMA, suggesting that mitogen-activated protein kinase (MAPK) cascade was involved downstream of the PKC pathway. In conclusion, the EGF/PKC/MAPK pathway regulates equine glycoprotein alpha subunit gene expression through a distinct regulatory region (-2300 to -1900) in trophoblasts, while essential elements for basal expression appear to exist within the -2800 to -1900 region of the promoter.

Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

Management of ankle sprains.

Without adequate care, acute ankle trauma can result in chronic joint instability. Use of a standardized protocol enhances the management of ankle sprains. In patients with grades I or II sprains, emphasis should be placed on accurate diagnosis, early use of RICE (rest, ice, compression and elevation), maintenance of range of motion and use of an ankle support. Sprains with complete ligament [corrected] tears (grade III) may require surgical intervention. Although early motion and mobility are recommended, ligamentous strength does not return until months after an ankle sprain.

Placental lactogen-I gene activation in differentiating trophoblast cells: extrinsic and intrinsic regulation involving mitogen-activated protein kinase signaling pathways.

Trophoblast giant cells are one of the primary endocrine cell types of the rodent placenta. Placental lactogen-I (PL-I) is the initial prolactin (PRL) family member expressed as trophoblast giant cells differentiate. In this report, we use the Rcho-1 trophoblast cell line as a model for studying the regulation of PL-I gene expression during trophoblast giant cell differentiation. Evidence is provided for trophoblast cell expression of epidermal growth factor receptor (EGFR), ErbB2, fibroblast growth factor receptor 1 (FGFR1), transforming growth factor-alpha, and heparin-binding EGF. EGF and FGF-2 stimulated PL-I mRNA and protein accumulation and PL-I promoter activity in a concentration-dependent manner. These latter growth factor actions on PL-I promoter activities were specifically inhibited by cotransfection with dominant negative constructs for EGFR and FGFRs respectively. Utilization of the mitogen-activated protein kinase (MAPK) pathway by EGF and FGF-2 in trophoblast cells was demonstrated by growth factor stimulation of a Gal4 DNA binding/Elk1 transactivational domain fusion construct, and more specifically by activation of extracellular signal regulated kinase and p38 MAPK. PL-I gene activation was also sensitive to disruption of MAPK and activation protein-1 (AP-1) signaling pathways. In conclusion, autocrine/paracrine pathways involving EGFR and FGFR1, MAPK and AP-1 are shown to participate in the regulation of the PL-I gene in differentiating trophoblast cells.

Patellar resurfacing in total knee arthroplasty.

Whether or not to resurface the patella when performing a primary total knee arthroplasty remains an open question. A number of recent studies have added new information relevant to this controversy. Anatomic studies show that there is normally substantial variability in the anatomy of the trochlear groove. Implanting a femoral component therefore results in a change in the surface topography of the knee in a high percentage of cases. Even though a number of intraoperative techniques have been described in an attempt to accurately reproduce femoral and tibial component rotation, studies of the application of these techniques reveal that component malpositioning or malrotation of a measurable degree occurs in 10% to 30% of cases, depending on the surgical technique and landmarks used. There has been substantial change in the design of both femoral and patellar components in recent years. Even with current designs, biomechanical studies indicate that some degree of change in kinematics and contact stresses occurs following total knee arthroplasty. However, the results of clinical studies have been extremely variable, with most showing either no difference or very little difference between resurfaced and nonresurfaced patellae in osteoarthritic knees. The decision to resurface the patella or not must be individualized on the basis of the surgeon's training and experience and an intraoperative assessment of the patellofemoral articulation.

P-glycoprotein levels predict poor outcome in patients with osteosarcoma.

To evaluate the relationship between the expression of P-glycoprotein by osteosarcomas and the rate of metastasis and death, a retrospective review of 172 patients who were diagnosed with osteosarcoma between 1987 and 1992 was performed. Forty patients had P-glycoprotein levels available. The majority of the osteosarcomas were Stage II-B (33 patients), with the remaining seven being Stage III. Tumor sites included 25 femurs, seven humeri, five tibias, and one each of pelvis, radius, and fibula. The patients with Stage III disease at presentation were treated differently from the time of diagnosis and therefore, these seven patients with Stage III osteosarcoma were excluded from additional analyses. The expression of P-glycoprotein by cultured tumor cells from biopsy specimens was determined using immunofluorescent microscopy. In the 33 patients with Stage IIB osteosarcoma with detectable P-glycoprotein, 67% (10 of 15) had metastases develop as compared with 28% (five of 18) of patients with undetectable P-glycoprotein. Similarly, 53% (eight of 15) of patients with tumors expressing P-glycoprotein died of disease compared with 11% (two of 18) with no detectable P-glycoprotein. Expression of P-glycoprotein by tumor cells seems to be associated with an estimated ninefold increase in the odds of death and a fivefold increase in the odds of metastases in patients with Stage IIB osteosarcoma. Kaplan-Meier survivorship analysis revealed that patients with detectable P-glycoprotein fared worse in terms of survival time and metastasis-free survival. Adjusting for covariates in the Cox proportional hazards model, expression of P-glycoprotein and its level were significantly predictive of time to death in patients with Stage IIB osteosarcoma.

Patient compliance and satisfaction with mechanical devices for preventing deep venous thrombosis after joint replacement.

A consecutive series of patients having total joint arthroplasty at a single university hospital were sequentially treated with two mechanical devices for prevention of deep venous thrombosis (DVT). The first 104 patients (group 1) wore thigh-high sequential compression device (SCD). The next 120 patients (group 2) wore a foot pump. Daily documentation of hourly compliance with each respective device was recorded until discharge. A patient satisfaction questionnaire was also obtained. Patient understanding about the devices' function aided compliance (73% compliance in group 1, and 77% in group 2). The satisfaction questionnaire revealed significantly greater satisfaction in group 2 (73%) versus group 1 (55%). Of a subgroup of 35 patients who had used both devices, 24 preferred the foot pump, 7 the SCD, and 4 had no preference. This study showed a higher degree of compliance and satisfaction for foot pumps as prophylaxis against DVT.

Improving readiness and fitness of the active military force through occupational medicine tenets.

The United States Military deploys its forces with minimal lead time. These forces must be medically qualified and physically fit for any locale and mission scenario. Historically, up to half of the force identified for deployment at any given time were not medically qualified. Matching individuals to specific occupations using validated medical and physical performance standards is an occupational medicine tenet that increases the effectiveness and efficiency of the workforce. To establish a cost-effective, valid medical program ensuring a fit and ready force, the military must: (1) develop validated physical fitness/occupational standards; (2) consolidate one fitness standard for males/females on the basis of workload requirements; (3) eliminate differing age standards; (4) provide statistically relevant medical screening, testing for health maintenance, and fitness for duty; and (5) mandate one joint medical standard for all military services.

Distinct regulatory regions from the prolactin-like protein C variant promoter direct trophoblast giant cell versus spongiotrophoblast cell-specific expression.

PRL-like protein C variant (PLP-Cv) is a newly identified member of the PRL family. PLP-Cv is specifically expressed in the chorioallantoic placenta by two distinct cell populations: trophoblast giant cells and spongiotrophoblast cells. To gain some insight regarding the control of PLP-Cv gene expression and the regulatory factors controlling trophoblast giant cell and spongiotrophoblast cell lineages, we have initiated a structural and functional analysis of the PLP-Cv promoter. The activities of a series of PLP-Cv promoter constructs, ranging in size from 4.5 kb to 50 bp, ligated to a luciferase reporter have been assessed in the Rcho-1 trophoblast cell line (restricted to trophoblast giant cell differentiation) and in a primary spongiotrophoblast cell culture system after transient transfection. PLP-Cv promoter constructs containing 4.5 kb to 149 bp of 5'-flanking DNA possessed full activity in the trophoblast giant cell model. A region located between -149 and -124 bp upstream of the PLP-Cv transcription start site was found to be essential for activation of the PLP-Cv promoter. Spongiotrophoblast cells required additional PLP-Cv 5'-flanking DNA for full activity. A region located between -2518 and -2242 bp upstream of the PLP-Cv transcription start site significantly enhanced PLP-Cv promoter in spongiotrophoblast cells. In conclusion, mechanisms underlying the activation of the PLP-Cv promoter are different in trophoblast giant cells vs. spongiotrophoblast cells.

The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.

Gonadotropin-releasing hormone activates the equine luteinizing hormone beta promoter through a protein kinase C/mitogen-activated protein kinase pathway.

GnRH regulation of LH secretion is well understood and involves Ca(2+) mobilization. However, the mechanism by which GnRH activates transcription of the LHbeta gene is controversial. GnRH is known to elevate intracellular calcium and activate the protein kinase C (PKC) pathway. The present study evaluated the pathway(s) involved in GnRH induction of LHbeta transcription. We have previously reported that the equine LHbeta (eLHbeta -448/+60) promoter is active in alphaT3-1 cells. Therefore, we created a clonal, stably transfected alphaT3-1 gonadotroph cell line harboring the eLHbeta promoter (-448/+60) fused to the luciferase reporter gene. Administration of a GnRH agonist resulted in induction of promoter activity that was completely inhibited by the antagonist antide. Various calcium-affecting drugs had no effect on the promoter. Administration of phorbol 12-myristate 13-acetate (PMA) elicited an activation similar to, albeit lower than, that with GnRH. Down-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated. Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, completely inhibited the activation of eLHbeta by PMA but only partly diminished GnRH's induction. Expression of the transcription factor, early growth response protein 1 (Egr1), correlated completely with activation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK acts. Our data suggest that GnRH induces activity of the eLHbeta promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.

Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression.

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family were found to induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contained two Egr response elements. Coexpression of Egr1 and SF-1 led to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuated this synergism. Endogenous expression of Egr1 in alpha T3-1 cells was not detectable under basal conditions, but was rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicated that these sites were required for GnRH induction. In fact, mutation of both Egr sites within the eLH beta promoter completely attenuated its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induced expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1- induced transcription of the eLH beta promoter.