Young and middle-aged female mice were ovariectomized and given cyclic injections of either estradiol or vehicle treatments. During the fifth week after surgery the Morris water maze was used to assess cognitive function. Age and treatment effects emerged over the course of spatial training such that middle-aged vehicle treated mice exhibited deficits in acquiring a spatial search strategy compared to younger vehicle treated mice and middle-age estradiol treated mice. Following behavioral characterization, mice were maintained on their injection schedule until week seven and hippocampi were collected 24 hr after the last injection. Hippocampal RNA was extracted and genes responsive to age and estrogen were identified using cDNA microarrays. Estradiol treatment in middle-aged mice altered the expression of genes related to transcriptional regulation, biosynthesis, growth, neuroprotection, and elements of cell signaling pathways. Expression profiles for representative genes were confirmed in a separate set of animals using oligonucleotide arrays and RT-PCR. Our results indicate that estrogen treatment in middle-aged animals may promote hippocampal health during the aging process.

The expression of the ERα and ERβ estrogen receptors in the hippocampus may be important in the etiology of age-related cognitive decline. To examine the role of ERα and ERβ in regulating transcription and learning, ovariectomized wild-type (WT) and ERα and ERβ knockout (KO) mice were used. Hippocampal gene transcription in young ERαKO mice was similar to WT mice 6 h after a single estradiol treatment. In middle-age ERαKO mice, hormone deprivation was associated with a decrease in the expression of select genes associated with the blood–brain barrier; cyclic estradiol treatment increased transcription of these select genes and improved learning in these mice. In contrast to ERαKO mice, ERβKO mice exhibited a basal hippocampal gene profile similar to WT mice treated with estradiol and, in the absence of estradiol treatment, young and middle-age ERβKO mice exhibited preserved learning on the water maze. The preserved memory performance of middle-age ERβKO mice could be reversed by lentiviral delivery of ERβ to the hippocampus. These results suggest that one function of ERβ is to regulate ERα-mediated transcription in the hippocampus. This model is supported by our observations that knockout of ERβ under conditions of low estradiol allowed ERα-mediated transcription. As estradiol levels increased in the absence of ERα, we observed that other mechanisms, likely including ERβ, regulated transcription and maintained hippocampal-dependent memory. Thus, our results indicate that ERα and ERβ interact with hormone levels to regulate transcription involved in maintaining hippocampal function during aging.

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.

This study used microarray analysis to examine age-related changes in gene expression 6 and 12 hr following a single estradiol injection in ovariectomized mice. Estradiol-responsive gene expression at the 6 hr time point was reduced in aged (18 mo) animals compared to young (4 mo) and middle-aged (MA, 12 mo) mice. Examination of gene clustering within biological and functional pathways indicated that young and MA mice exhibited increased expression of genes for cellular components of the synapse and decreased expression of genes related to oxidative phosphorylation and mitochondrial dysfunction. At the 12 hr time point, estradiol-responsive gene expression increased in aged animals and decreased in young and MA mice compared to the 6 hr time point. Gene clustering analysis indicated that aged mice exhibited increased expression of genes for signaling pathways that are rapidly influenced by estradiol. The age differences in gene expression for rapid signaling pathways may relate to disparity in basal pathway activity and estradiol mediated activation of rapid signaling cascades.

Hepatocyte growth factor (HGF) is a paracrine factor involved in organogenesis, tissue repair, and wound healing. We report here that HGF promotes osteogenic differentiation through the transcription of key osteogenic markers, including osteocalcin, osterix, and osteoprotegerin in human mesenchymal stem cells and is a necessary component for the establishment of osteoblast mineralization. Blocking endogenous HGF using PHA665752, a c-Met inhibitor (the HGF receptor), or an HGF-neutralizing antibody attenuates mineralization, and PHA665752 markedly reduced alkaline phosphatase activity. Moreover, we report that HGF promotion of osteogenic differentiation involves the rapid phosphorylation of p38 and differential regulation of its isoforms, p38α and p38β. Western blot analysis revealed a significantly increased level of p38α and p38β protein, and reverse transcription quantitative PCR revealed that HGF increased the transcriptional level of both p38α and p38β. Using small interfering RNA to reduce the transcription of p38α and p38β, we saw differential roles for p38α and p38β on the HGF-induced expression of key osteogenic markers. In summary, our data demonstrate the importance of p38 signaling in HGF regulation of osteogenic differentiation.