Supplementary MaterialsFigure S1: Confirmation of the effect of ovariectomy and estrogen
Supplementary MaterialsFigure S1: Confirmation of the effect of ovariectomy and estrogen supplementation. of conversion from membrane-bound Sema4D to secreted Sem4D was significantly higher at the time of vaginal opening than at any other developmental stage. Open in a separate window Figure 1 Increase of soluble Sema4D and reorganization of Plexin-B1 receptor during vaginal development.(A) The membrane-bound (larger) and soluble (smaller) forms of Sema4D are evident on western blots containing proteins from vaginal tissue extracts. The precursor (larger) and active form (smaller) of Plexin-B1 are also evident on western blots containing proteins from vaginal tissue extracts; the active 75 kDa form represents a fragment of the 300 kDa Plexin-B1 precursor produced by proprotein convertase. The fragments resulting from the proprotein convertase-dependent cleavage are integrated into signaling-active receptors, which have a distinct conformation from the precursors . Age (weeks): vaginal protein extracts from 2-, 3-, 4-, 4.5-, 5-, 8-, 9-, or 18-week-old mice. Mb: membrane-bound. (B) The ratio of soluble Sema4D to total Sema4D increases significantly in 5-week-old mouse vaginal tissue. Each data point represents the mean SEM of 3 to 6 mice. *mRNA level was also significantly higher in 5-week-old vagina tissue based on real-time RT-PCR analysis (Fig. 1E). Reorganization of Plexin-B1 during Vaginal Development Western blots were probed with anti-Plexin-B1 antibodies to examine whether the expression of the Sema4D receptor, Plexin-B1, changed during vaginal development. Vaginal tissue protein extracts from each developmental stage and anti-Plexin-B1 antibodies that detected both a 300 kDa form and a smaller 75 kDa form were used for this analysis (Fig. 1A). The 300 kDa protein represents the precursor form of Plexin-B1 that exists prior to digestion by a protease, proprotein convertase . The smaller 75 kDa protein VX-765 distributor represents the Plexin-B1 fragment that is generated by proprotein convertase-mediated digestion . After convertase-mediated digestion of the Plexin-B1 precursor, the conformation of Plexin-B1 protein structure is transformed into VX-765 distributor an active form that transmits Sema4D Rabbit Polyclonal to LAT signal more intensely (20, Fig. S2). Interestingly, the ratio of the smaller 75 kDa band to the VX-765 distributor -actin band was significantly higher for the 5-week sample than for any other sample (Fig. 1F). Thus the western blot findings indicated that the active form of Plexin-B1 was highest at 5 weeks, which is the time of mouse vaginal opening. Real-time PCR analysis demonstrated that mRNA levels were constant during postnatal vaginal development except that the mRNA declined significantly by the 11th week (Fig. 1E). Conversion of Sema4D and of Plexin-B1 is Estrogen-dependent Postnatal vaginal remodeling in mice is a hormonally triggered process ; therefore, we investigated whether proteolytic release of Sema4D and reorganization of Plexin-B1 during remodeling were each estrogen dependent. Mice were initially subjected to ovariectomy on postnatal day 22; one third of these mice were VX-765 distributor each injected with exogenous estrogen (OVX-E2 mice), another third with vehicle only (OVX-oil mice, Fig. 2A) and the remaining third received no injection (OVX mice). Western blot analysis was used to examine both Sema4D proteolysis and Plexin-B1 conversion in these ovariectomized mice when the mice became 5 weeks old (Fig. 2B). Conversion of membrane-bound Sema4D into the soluble form was significantly lower in OVX mice than in sham-operated WT mice (Fig. 2B, C). In contrast, this Sema4D conversion was significantly higher in WT OVX-E2 than in WT OVX-oil mice (Fig. 2B, C); these findings indicated that proteolytic conversion of Sema4D was estrogen dependent. Reorganization of Plexin-B1 into an active form was significantly lower in WT OVX mice than in sham-operated WT mice (Fig. 2B, D). The reorganization of Plexin-B1 was significantly higher in WT OVX-E2 mice than in WT OVX-oil mice; these findings indicated Plexin-B1 reorganization during mouse vaginal tissue remodeling was estrogen dependent (Fig..