Fos proteins are not prerequisite for osmotic induction of vasopressin transcription in supraoptic nucleus of rats

Abstract

While it is well known that osmotic stimulation induces the expression of Fos family members in the supraoptic nucleus (SON), it is unclear whether the induced protein products are involved in the regulation of the gene transcription of arginine vasopressin (AVP). In the present study, we examined the in vivo correlation between changes in AVP gene transcription and expression of the various Fos family members in the SON after acute osmotic stimuli. The data demonstrated that the peak of AVP transcription (measured by intronic in situ hybridization) observed 15min after an injection of hypertonic saline preceded the expression of Fos proteins, which became detectable at 30min and peaked at 120min. Electrophoretic mobility shift assay showed that the expressed Fos proteins bound to the composite AP-1/CRE-like site in the AVP promoter. These data suggest that Fos proteins in the SON induced by acute osmotic stimuli could affect AVP gene transcription by binding to the AVP promoter, but they are not prerequisite for the induction of AVP gene transcription.

Figure 1

Time course of expression of Fos family mRNA in the SON after hypertonic saline (HS) injection. Rats were decapitated at 15, 30, 60, 120, 240, 360 and 720 min after an ip injection of HS, or 120 min after isotonic saline (IS) injection. Bars represent the mean ± S.E. (arbitrary units) of the values obtained from 6 rats in each experimental group. Representative images of SON in rats injected with IS and HS (60 min after injection for c-fos, 120 min ater injection for fra-1, fra-2 and fosB) are also shown. *, p<0.05 versus control.

Figure 2

Time course of expression of Fos family proteins relative to AVP gene transcription in the SON after HS injection. A. Western blot analysis performed with pan-Fos antibody on whole cell protein extracts from pools of SON punches collected at the indicated times from rats without treatment or injected with either isotonic saline (IS) or hypertonic saline (HS). B. Quantitative in situ analysis of AVP hnRNA levels in the SON of rats without treatment or injected with either IS or HS. Bars represent the mean ± S.E. arbitrary units of the values obtained from 6 rats in each experimental group. Representative images of AVP hnRNA expression at each time point are also shown. *, p<0.05 versus values at 60 min.

Figure 3

AP-1 electrophoretic mobility gel shift assay (EMSA) with antibodies for Fos members. EMSA was performed in the presence of either a pan-Fos antibody or specific antibodies raised against the c-Fos, Fra-1, Fra-2 or FosB, and using proteins extracted from the SON of rats injected with either isotonic saline (control) or hypertonic saline (120, 240 or 360 min) and a ³²P-radiolabeled probe containing either a classical AP-1 consensus element (A) or the AVP derived, 37-bp long sequence containing both the AP-1 and CRE-like sites (B). The figure is representative of 3 experiments. The arrow indicates specific AP-1 binding, of which intensity was quantified and shown as bar graphs. The levels of AP1 intensity in control at each time point are shown as dotted lines. *; supershifted bands in pan-Fos and Fra-2 lanes. ns: non-specific bands shown in only lanes where serum was applied instead of IgG.