Regulation of Corticotropin-Releasing Factor-Binding Protein Expression in Amygdalar Neuronal Cultures

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73102/1/j.1365-2826.1999.00413.x.pd

Exposure to an Antisense Oligonucleotide Decreases Corticotropin‐Releasing Factor Receptor Binding in Rat Pituitary Cultures

: Corticotropin‐releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of “antisense knockdown” strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15‐mer antisense (5′ CTG‐CGG‐GCG‐CCG‐TCC 3′) and “scrambled” control (5′ CGT‐CCG‐CGC‐GCT‐GCG 3′) oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in 125I‐ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of “scrambled” oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30‐min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS

Corticotropin-releasing factor is secreted in the BE(2)-C neuroblastoma cell and is responsive to forskolin

The BE(2)-C human neuroblastoma has been shown to express corticotropin-releasing factor (CRF) messenger RNA. In this study, BE(2)-C cells were treated 5 days with 5 μM retinoic acid. Cell extracts were also applied onto a C18 Vydac column and fractions were assayed for CRF-like immunoreactivity (CRF-LI) which coincided in time of elution with oxidized or non-oxidized CRF synthetic CRF standard. With forskolin treatment, secretion media and cell extract CRF-LI increased in a concentration-dependent fashion. Thus, this cell line synthesizes and secretes CRF and is a good model for studying CRF regulation

The BE (2)-M17 neuroblastoma cell line synthesizes and secretes corticotropin-releasing factor

The BE (2)-M17 human neuroblastoma has previously been show to express corticotropin-releasing factor (CRF) mRNA following retinoic acid treatment. It is demonstrated in this report that both cell extracts and cell incubation medium of retinoic acid-treated BE (2)-M17 cells were shown to contain CRF-like immunoreactivity (CRF-LI) by RIA. CRF-LI secretion and content were also dose-dependently increased by forskolin. In addition, cell extracts were applied to a C 18 Vydac column and peak CRF-LI from the collected fractions was shown to coincide in time of elution with peak immunoreactivity seen with oxidized synthetic CRF standard. Thus, in containing the CRF peptide, the BE (2)-M17 cells are useful models for further study of CRF cellular and genetic regulation

Regulation of Corticotropin‐Releasing Factor Secretion and Synthesis in the Human Neuroblastoma Clones‐ BE(2)‐M17 and BE(2)‐C

The BE(2)‐M17 and BE(2)‐C human neuroblastoma cell lines have been shown to synthesize and secrete corticotropin‐releasing factor (CRF) following retinoic acid treatment. It has been demonstrated that CRF secretion and intracellular synthesis increases in response to forskolin treatment. In this report, we have further characterized these cells in response to protein kinase C activators, dexamethasone, interleukin—1x, as well as various neurotransmitters and peptides. Nanomolar concentrations of the phorbol ester—phorbol 12 myristate 13—acetate (TPA), increased intracellular CRF content in both cell lines while increasing secretion only in the BE(2)‐M17 cell. Nanomolar concentrations of dexamethasone were not able to alter basal levels of secretion and content in either cell type. However, in the BE(2)‐Ml7 cell but not the BE(2)‐C cell, the same concentrations of dexamethasone added to 30 μM forskolin augmented levels of CRF secretion and content. Likewise, the same augmented response in CRF secretion and content was seen only in the BE(2)‐M17 cell line when nanomolar concentrations of dexamethasone were added to 20 nM TPA. Furthermore, only in the BE(2)‐M17 cell line were micromolar levels of the biogenic amine serotonin able to increase levels of CRF secretion and content. No effects on CRF in both cell lines were demonstrable with picomolar levels of interleukin‐10: as well as micromolar levels of acetylcholine, norepinephrine, arginine‐vasopressin, oxytocin, and angiotensin‐II. The potential usefulness of these cells as models of central nervous system or placental CRF‐containing neurons is discussed