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Fig. 3. Estrogen up-regulation of trkA mRNA in PC12 cells. Northern blot analysis of PC12 mRNA probed for trkA. Top: Total RNA (20 mg) was size-fractionated on a 1.2% agarose gel and transferred to nylon. Blots were hybridized to a random-primed (32P) cDNA probe of approximately 0.5 kb (gift of Dr. Luis F. Parada) for 48 h, washed at high stringency (0.2 x SSC, 50┬░C, 2 h) and apposed to film (Kodak X-RP). PC12 cells were exposed to either NGF (100 ng/mL) alone for 18 d or to NGF alone for 10 days, followed by NGF plus estrogen (10-9 M) concurrently for an additional 8 days. Bottom: Same blot probed for P-actin mRNA, using a 40 base oligonucleotide (Dupont), 3'-labeled with 32P, using terminal deoxynucleotidyl transferase. Densitometric measurements, normalized to P-actin, suggest that, in the presence of NGF, estrogen appears to increase trkA mRNA expression significantly. (Adapted with permission from ref. 14).

may regulate neurite growth and differentiation by interactions with neurotrophins through autocrine or local paracrine mechanisms.

Estrogen and the neurotrophins may influence each other's actions to regulate receptor and/or ligand availability by actions at the transcriptional, translational, and posttransla-tional levels. In this regard, differential and reciprocal regulation of estrogen and NGF receptor mRNAs (both p75 and trkA) by their ligands has been shown in both adult sensory neurons (24) and PC12 cells (23). In both PC12 cells (Fig. 3) and following estrogen treatment of the ovariectomized adult female rat, p75 mRNA was transiently down-regulated; whereas trkA mRNA expression was significantly up-regulated and sustained. NGF was also found to up-regulate estrogen binding in both PC12 cells (23) and cerebral cortical explants (80) (Fig. 4). Unlike the pattern seen in PC12 cells, however, there was no apparent associated change in cortical ER mRNA expression.

Interactions of estrogen and the neurotrophins may also be involved in the as yet unknown mechanisms that underlie differential regulation by estrogen of its own receptor in the brain. The direction of the responses of the neural ER to estrogen appears to be developmental-stage dependent. Thus, although estrogen classically down-regulates its receptor in the adult brain (102,103), a considerable degree of ER mRNA expression is seen in the developing postnatal brain until around postnatal d 28 (22), despite postnatal levels of estrogen normally sufficient for receptor down-regulation in the adult. One way of interpreting such regulatory patterns would be to consider that they may result, during the postnatal period, from either estrogen up-regulation of its receptor or from the inability of estrogen to regulate its own receptor (nonregulation) (14). One might further speculate that developmental stage-dependent differences in the direction of estrogen

Cerebral Cortex Septum

Fig. 4. NGF regulates estrogen binding by the unliganded estrogen receptor. Specific, nuclear [3H]moxestrol binding (normalized to DNA content) was observed in both postnatal d 2 (P2) cortical and basal forebrain cultured explant slices, maintained for 8 d in vitro in the absence of estradiol. The addition of human recombinant NGF led to a significant increase (**) in nuclear estrogen binding sites in cortical but not basal forebrain explants. NGF regulation of estrogen binding under estrogen-deficient conditions appears to be region-specific in the explants. This apparent specificity, however, may reflect differences in the ontogeny of estrogen binding in these regions. Cortical receptors develop significantly earlier than those of the basal forebrain. Maximal receptor levels are reached ~P8-10 in the cortex; ~puberty in the basal forebrain (D. Toran-Allerand, unpublished observations). n = sample size in each group. (Adapted with permission from ref. 79.)

Cerebral Cortex Septum

Fig. 4. NGF regulates estrogen binding by the unliganded estrogen receptor. Specific, nuclear [3H]moxestrol binding (normalized to DNA content) was observed in both postnatal d 2 (P2) cortical and basal forebrain cultured explant slices, maintained for 8 d in vitro in the absence of estradiol. The addition of human recombinant NGF led to a significant increase (**) in nuclear estrogen binding sites in cortical but not basal forebrain explants. NGF regulation of estrogen binding under estrogen-deficient conditions appears to be region-specific in the explants. This apparent specificity, however, may reflect differences in the ontogeny of estrogen binding in these regions. Cortical receptors develop significantly earlier than those of the basal forebrain. Maximal receptor levels are reached ~P8-10 in the cortex; ~puberty in the basal forebrain (D. Toran-Allerand, unpublished observations). n = sample size in each group. (Adapted with permission from ref. 79.)

regulation of its own receptor may perhaps be a consequence of interactions with other transcription-regulating molecules, including the neurotrophins. To explain this discrepancy in the responses of the ER during development and in the adult, I have proposed that the neurotrophins may serve as regulatory switches whereby their modulatory role on ER expression may influence the direction of estrogen regulation of its own receptor (Fig. 5) (14,15). For example, during CNS development, the ability of the neurotrophins to increase ER protein levels and binding significantly may be sufficient alone (or even in synergy with estrogen) to influence or even override the intrinsic suppressive action of estrogen on its own receptor. Maturation of the CNS is associated with significant alterations in the spatial, temporal, and functional expression of the neurotrophins and their receptors (104,105), as well as in their physiological role(s) (106). Such changes may serve to "free" the ER from the regulatory influences of the neurotrophins, resulting in the emergence of the intrinsic (adult) pattern of estrogen-induced receptor down-regulation and loss of estrogen's neurite-promoting effects. However, in the adult, following injury to estrogen target regions or in the presence of estrogen deficiency (e.g., ovariectomy, menopause), there may be a "switch" in the direction of estrogen regulation of its receptor back to the developmental pattern that is manifested by a re-expression of the growth-promoting properties of estrogen which is never seen in the normal adult. Because various forms of trauma and injury to the brain (perhaps including steroid deficiency) also

Development / "Injured" Adult Normal Adult

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