This study evaluates the transcriptional and post-transcriptional regulation of prolactin (PRL) by vasoactive intestinal peptide (VIP). Pituitary nuclei from laying (control), incubating (with enhanced VIP secretion), and VIP-immunized laying turkey hens, and from pituitary cells cultured with or without VIP were used in nuclear run-on transcription assays. Cytoplasmic PRL mRNA was analyzed by slot blot hybridization. PRL transcription was greater in hyperprolactinemic incubating birds (PRL/beta-actin=3.33) than in laying birds (PRL/beta-actin=1.83). VIP-immunoneutralized birds had 47% and 51% decreases in PRL transcription and cytoplasmic PRL mRNA, respectively when compared with laying birds. In primary pituitary cell cultures, VIP significantly increased the transcription rate of PRL (3.8-fold) and cytoplasmic PRL mRNA (3.2-fold) compared with that of non-VIP-treated pituitary cells. The stability of pre-existing PRL mRNA was measured by Northern blot analysis after addition of actinomycin D. PRL mRNA half-lives were calculated using a two-component model, with a first-long component of 18.0+/-1.0 h and a second-short component of 3.7+/-0.7 h in non-VIP-treated pituitary cells. Both half-lives were significantly increased (53. 2+/-6.9 and 26.3+/-4.3 h) in VIP-treated cells. The present data show that VIP acts to stimulate PRL expression by up-regulating the transcription rate of PRL and by enhancing PRL mRNA stability.
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Z Tong, GR Pitts, S You, DN Foster, and ME El Halawani
S You, L K Foster, J L Silsby, M E El Halawani, and D N Foster
ABSTRACT
cDNAs encoding the precursor molecule of the turkey LH β subunit (tLHβ) were cloned from a turkey pituitary cDNA library. The nucleotide sequence of the longest of two different tLHβ cDNA clones contained 592 bp, and included 23 bp of the 5′ untranslated region (UTR) and 92 bp of the 3′ UTR in addition to a 477 bp open reading frame that encoded a 39 amino acid leader polypeptide and a 120 amino acid mature apoprotein. Turkey and chicken LHβ sequences shared approximately 92 and 93% nucleotide and amino acid sequence similarities respectively. Northern blot analysis of total cellular anterior pituitary RNA showed that an approximate 800 base transcript hybridized to a 32P-labelled tLHβ cDNA probe.
The gonadotrophin-releasing hormone (GnRH)-and prolactin (PRL)-regulated expression of LH and PRL in dispersed pituitary cells was determined by Northern blot analysis of tLHβ and PRL steady-state mRNA levels and by RIA analysis of secreted LH and PRL. GnRH-treated cells showed increased levels of both tLHβ mRNA and secreted LH, whereas mRNA and secreted levels of PRL did not change significantly. Cells treated with PRL showed lower levels of tLHβ and PRL mRNA as well as decreased release of LH and PRL. When cells were treated with both PRL and GnRH, increases in tLHβ mRNA and secreted levels of LH observed with GnRH alone were negated, whereas the decreases in mRNA and secreted levels of PRL observed with PRL alone were abrogated.
These findings suggest that PRL can down-regulate tLHβ gene expression and spontaneous release of LH as well as autoregulate PRL gene expression and spontaneous release of PRL, while GnRH appears capable of modulating the effects of PRL-regulated LH and PRL gene expression and spontaneous release.
S Park, W Lee, KH You, H Kim, JM Suh, HK Chung, M Shong, and OY Kwon
This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.
