Substance P (SP) (a G protein protein-coupled receptor neuropeptide) and 17-estradiol (E2) (a nuclear receptor sex steroid hormone) are important in cellular biology. SP regulates cellular processes in the CMS, placenta and vasculature including permeability, angiogenesis, mitogenesis, and transformation. Examples of sexual dimorphism in cellular function suggest genetic hormonal control. Using Northern blot analysis of SPR mRNA levels we studied the effects of E2 on SPR gene expression in AR42 j (rat pancreatic acinar) cells which constitutively express high levels of SPR. E2 was incubated with AR42J cells in low hormone serum for 0, '/S, 1, 2, and 4 hrs Total RNAwas isolated from cells using phenol chloroform extraction and poly A selected with oligo (dT) columns. Samples were run on formaldehyde gels, transferred to nylon membranes and hybridized with [alz-P] dCTP labelled random pnmer cDNA probes at high stringency. Radiographs were developed after 1-3 days. The level of SPR mRNA was normalized to a marker cDNA for Chinese hamster ovary cells by a ratio of the 2 densitometry signals expressed as percent change from control (E, at time 0). E, (100nM) led to an 89% increase in SPR mRNA levels (4.7 kb band) and the appearance of a novel approx. 2 kb band suggesting a possible alternatively spliced variant. The effect was greatest after 1-3 hours and was concentration (0.1-1000nM) dependent with a peak response at 100nM E2 . The response was inhibited by the RNA polymerase inhibitor actinomycin D (5 ug/ml) but not by the translational inhibitor cycloheximide (10 ug/ml) In addition, the antiestrogen tamoxifen (1 uM) blocked the stimulatory effect of E2 on SPR mRNA. In conclusion, our studies indicate that E2 stimulates SPR gene expression,.that the control is at the level of transcription and involves interaction with the E2R. This study has implications for hormonal control of vasoactive peptide gene expression.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology