Histone deacetylase inhibitors enhance the apoptotic activity of insulin-like growth factor binding protein-3 by blocking PKC-induced IGFBP-3 degradation

Abstract The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased ∼3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3′-untranslated region (3′-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

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Oestrogen regulation of insulin-like growth factor binding protein-3 (IGFBP-3) and expression of IGFBP-3 messenger RNA in the ovine endometrium

Reproduction Fertility and Development ◽

10.1071/r97082 ◽

1998 ◽

Vol 10 (3) ◽

pp. 241 ◽

Cited By ~ 9

Author(s):

A. J. Peterson ◽

A. M. Ledgard ◽

S. C. Hodgkinson

Keyword(s):

Growth Factor ◽

Binding Protein ◽

Oestrous Cycle ◽

Endometrial Tissue ◽

Northern Analysis ◽

Northern Blot Hybridization ◽

Insulin Like Growth Factor ◽

Factor Binding ◽

Oestradiol Treatment ◽

Igfbp 3

Transcriptional regulation of insulin-like growth factor binding protein-3 (IGFBP-3) by oestrogen was investigated by Northern blot hybridization of endometrial tissue mRNA from anoestrous ewes treated with oestradiol-17β at either 0, 40 or 80 µg per day for 7 days. The 2.6 kb IGFBP-3 transcript, seen in control animals, was virtually undetectable in treated animals. This suppressive effect was reflected in Western ligand blots of the uterine luminal fluid (ULF) proteins where the concentration of IGFBP-3 was significantly decreased with increasing oestrogen treatments. IGFBP-2 levels were increased with oestradiol treatment but no significant effect was seen on the other minor IGFBP’s present in the ULF. Northern analysis also showed that the IGFBP-3 transcript was present from days 12 to 16 of the oestrous cycle but was either absent or very weak on days 0 (oestrus) and 9 of cycling ewes. In situ hybridization of endometrial tissue sections localized the IGFBP-3 mRNA to the luminal epithelial cell layer, areas of the stromal tissue and in some glandular epithelial cells. Oestradiol treatment of ewes down-regulated expression of IGFBP-3 in the endometrium; therefore, the low levels of IGFBP-3 in ULF during the early part of the oestrous cycle is possibly due to elevated levels of plasma oestradiol around oestrus.

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