Kevin W. Aylor

Gonadotropin-Releasing Hormone Regulation of Gonadotropin Subunit Gene Expression in Female Rats: Actions on Follicle-Stimulating Hormoneβ Messenger Ribonucleic Acid (mRNA) Involve Differential Expression of Pituitary Activin (β-B) and Follistatin mRNAs1

GnRH is the primary stimulus in the regulation of gonadotropin subunit mRNA expression. Additionally, local (pituitary) production of activin and follistatin appear to modulate the expression of FSH β mRNA. The current studies aimed to determine whether GnRH regulation of pituitary activin (β-B) and follistatin mRNAs could play a role in the differential actions of GnRH pulse pattern on gonadotropin mRNA expression in female rats. In response to altered GnRH pulse amplitude, the expression of FSH β and follistatin mRNAs followed an inverse pattern. Only high dose GnRH increased expression of follistatin whereas, in contrast, β-B and FSH β expression were increased following lower doses of GnRH. To determine whether increased follistatin mRNA expression was correlated with FSH β mRNA responses, we examined their temporal relationship following high dose GnRH. Both FSH β and follistatin mRNAs were increased within 2 h and remained increased through 6 h. However, by 12 h FSH β mRNA levels returned to values ...

Regulation of Intracellular Signaling Cascades by GNRH Pulse Frequency in the Rat Pituitary: Roles for CaMK II, ERK, and JNK Activation

Abstract Pulsatile GnRH (GNRH) differentially regulates LH and FSH subunit genes, with faster frequencies favoring Lhb transcription and slower favoring Fshb. Various intracellular pathways mediate the effects of GNRH, including CaMK II (CAMK2), ERK, and JNK. We examined whether activation of these pathways is regulated by GNRH pulse frequency in vivo. GNRH-deficient rats received GNRH pulses (25 ng i.v. every 30 or 240 min for 8 h, vehicle to controls). Pituitaries were collected 5 min after the last pulse, bisected, and one half processed for RNA (to measure beta subunit primary transcripts [PTs]) and the other for protein. Phosphorylated CAMK2 (phospho-CAMK2), ERK (mitogen-activated protein kinase 1/3 [MAPK1/3], also known as p42 ERK2 and p44 ERK1, respectively), and JNK (MAPK8/9, also known as p46 JNK1 and p54 JNK2, respectively) were determined by Western blotting. The 30-min pulses maximally stimulated Lhb PT (8-fold), whereas 240 min was optimal for Fshb PT (3-fold increase). Both GNRH pulse frequencies increased phospho-CAMK2 4-fold. Activation of MAPK1/3 was stimulated by both 30- and 240-min pulses, but phosphorylation of MAPK3 was significantly greater following slower GNRH pulses (240 min: 4-fold, 30 min: 2-fold). MAPK8/9 activation was unchanged by pulsatile GNRH in this paradigm, but as previous results showed that GNRH-induced activation of MAPK8/9 is delayed, 5 min after GNRH may not be optimal to observe MAPK8/9 activation. These data show that CAMK2 is activated by GNRH, but not in a frequency-dependant manner, whereas MAPK3 is maximally stimulated by slow-frequency GNRH pulses. Thus, the ERK response to slow pulse frequency is part of the mechanisms mediating Fhb transcriptional responses to GNRH..

Gonadotropin Subunit Transcriptional Responses to Calcium Signals in the Rat: Evidence for Regulation by Pulse Frequency

Abstract Alterations in the frequency of calcium influx signals to rat pituitary cells can regulate the expression of gonadotropin subunit mRNAs in a differential manner, producing effects that are similar to those previously found for GnRH. The present study was conducted to investigate whether this reflects a transcriptional response to calcium pulse frequency, as determined by alterations in primary transcript (PT) expression. Perifused rat pituitary cells were given pulses of the calcium channel-activator Bay K 8644 (BK; with 10 mM KCl in the injectate) for 6 h. The response to alterations in pulse dose was examined by giving pulses of 1, 3, or 10 μM BK at 60-min intervals. Maximal increases in LHβ and FSHβ PTs were obtained with the 3-μM BK pulse dose and with the 10-μM dose for α. To investigate the effect of calcium pulse frequency, 3-μM BK pulses were given at intervals of 15, 60, or 180 min. Alpha PT was selectively stimulated by 15-min pulses and LHβ by 15- and 60-min pulses of BK. In contrast, FSHβ PT was maximally stimulated by the slower, 180-min pulse interval. These findings reveal that pulsatile increases in intracellular calcium stimulate α, LHβ, and FSHβ transcription in a differential manner. Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH pulse signals from the plasma membrane to the gene, and they may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.

Regulation of Lhb and Egr1 Gene Expression by GNRH Pulses in Rat Pituitaries Is Both c-Jun N-Terminal Kinase (JNK)- and Extracellular Signal-Regulated Kinase (ERK)-Dependent

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1–24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.

Pituitary Follistatin Gene Expression in Female Rats: Evidence That Inhibin Regulates Transcription

Abstract Follistatin (FS), along with the members of the transforming growth factor β family activin and inhibin, are important regulators of FSH secretion and messenger RNA production. While activin and inhibin appear to function as tonic modulators of FSH (stimulatory and inhibitory, respectively), dynamic changes in FS are noted through the estrous cycle and under varying physiological experimental paradigms. This suggests that FS is a major contributor to the precisely coordinated secretion of FSH that maintains reproductive function. The aim of this study was to investigate changes in FS, in particular the early (<12 h) rise observed after ovariectomy (OVX), and to determine whether these changes were as a consequence of variations in gene transcription rates. FS primary transcript (PT) and mRNA were found to increase 3-fold 12 h post-OVX, indicating increased gene transcription during this time period. Replacement with estradiol and/or blockade of GnRH had only modest effects on FS PT concentration. Inhibin immunoneutralization of intact rats resulted in a 3-fold increase in FS PT 12 h after administration of inhibin α antisera. Significant increases in FS mRNA at both 2 and 12 h also suggested that inhibin also may have effects on message stability. After administration of recombinant human inhibin A, there was a prompt decline in both FS PT and mRNA. These results indicate that inhibin is a major regulator of FS, both by transcriptional and nontranscriptional mechanisms.

Pathways for insulin access to the brain: the role of the microvascular endothelial cell

Insulin affects multiple important central nervous system (CNS) functions including memory and appetite, yet the pathway(s) by which insulin reaches brain interstitial fluid (bISF) has not been clarified. Recent studies demonstrate that to reach bISF, subarachnoid cerebrospinal fluid (CSF) courses through the Virchow-Robin space (VRS) which sheaths penetrating pial vessels down to the capillary level. Whether insulin predominantly enters the VRS and bISF by local transport through the blood-brain barrier, or by being secreted into the CSF by the choroid plexus, is unknown. We injected 125I-TyrA14-insulin or regular insulin intravenously and compared the rates of insulin reaching subarachnoid CSF with its plasma clearance by brain tissue samples (an index of microvascular endothelial cell binding/uptake/transport). The latter process was more than 40-fold more rapid. We then showed that selective insulin receptor blockade or 4 wk of high-fat feeding each inhibited microvascular brain 125I-TyrA14-insulin clearance. We further confirmed that 125I-TyrA14-insulin was internalized by brain microvascular endothelial cells, indicating that the in vivo tissue association reflected cellular transport, not simply microvascular tracer binding.

Testosterone Stimulates Follicle-Stimulating Hormone β Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells

Abstract This study investigated whether estradiol (E2) or testosterone (T) activate extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinase II (Ca/CaMK II), as indicated by enzyme phosphorylation in rat pituitaries. In vivo studies used adult female rats given E2, T, or empty silastic capsules (vehicle controls). Twenty-four hours later, the rats were given a single pulse of GnRH (300 ng) or BSA-saline (to controls) and killed 5 min later. GnRH stimulated a two- to three-fold rise in activated Ca/CaMK II, and E2 and T had no effect on Ca/CaMK II activation. In contrast, both GnRH and T stimulated threefold increases in ERK activity, with additive effects seen following the combination of GnRH+T. E2 had no effect on ERK activity. In αT3 clonal gonadotrope cells, dihydrotestosterone did not activate ERK alone but enhanced and prolonged the ERK responses to GnRH, demonstrating direct effects on the gonadotrope. Thus, the ERK response to GnRH plus androgen was enhanced in both rat pituitary and αT3 cells. In vitro studies with cultured rat pituitary cells examined the effect of GnRH±T in the presence of the mitogen-activated protein (MAP) kinase kinase inhibitor, PD-098059 (PD). Results showed that PD suppressed ERK activational and FSHβ transcriptional responses to T. These findings suggest that one site of T regulation of FSHβ transcription is through the selective stimulation of the ERK pathway.

Unravelling the regulation of insulin transport across the brain endothelial cell

Aims/hypothesisFor circulating insulin to act on the brain it must cross the blood–brain barrier (BBB). Remarkably little is known about how circulating insulin crosses the BBB’s highly restrictive brain endothelial cells (BECs). Therefore, we examined potential mechanisms regulating BEC insulin uptake, signalling and degradation during BEC transcytosis, and how transport is affected by a high-fat diet (HFD) and by astrocyte activity.Methods125I–TyrA14-insulin uptake and transcytosis, and the effects of insulin receptor (IR) blockade, inhibition of insulin signalling, astrocyte stimulation and an HFD were tested using purified isolated BECs (iBECs) in monoculture and co-cultured with astrocytes.ResultsAt physiological insulin concentrations, the IR, not the IGF-1 receptor, facilitated BEC insulin uptake, which required lipid raft-mediated endocytosis, but did not require insulin action on phosphoinositide-3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK). Feeding rats an HFD for 4xa0weeks decreased iBEC insulin uptake and increased NF-κB binding activity without affecting insulin PI3K signalling, IR expression or content, or insulin degrading enzyme expression. Using an in vitro BBB (co-culture of iBECs and astrocytes), we found insulin was not degraded during transcytosis, and that stimulating astrocytes with l-glutamate increased transcytosis, while inhibiting nitric oxide synthase decreased insulin transcytosis.Conclusions/interpretationInsulin crosses the BBB intact via an IR-specific, vesicle-mediated transport process in the BECs. HFD feeding, nitric oxide inhibition and astrocyte stimulation can regulate BEC insulin uptake and transcytosis.

Stimulation of FSHβ transcription by blockade of endogenous pituitary follistatin production

This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript and FSH release, and responses were doubled in the presence of adenovirus- FST-AS. These results demonstrate the effectiveness of adenovirus FST-AS in suppressing pituitary FST protein expression and enhancing FSH biological responses at the transcriptional level. Thus, the FST-deficient rat gonadotrope cell is a model that allows for the investigation of factors regulating FSHbeta expression, which might otherwise involve the autocrine/paracrine actions of FST.This study investigated FSHβ transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85–90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1–3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus—AS construct. Infection with adenovirus—FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHβ primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHβ primary transcript and FSH release, and responses were doubled in the presence of adenovirus—FST-AS. These results demonstrate the effectiveness of adenovirus FST-AS in suppressing pituitary FST protein expression and enhancing FSH biological responses at the transcriptional level. Thus, the FST-deficient rat gonadotrope cell is a model that allows for the investigation of factors regulating FSHβ expression, which might otherwise involve the autocrine/paracrine actions of FST.