Farhan A. Syed

Skeletal Effects of Estrogen Are Mediated by Opposing Actions of Classical and Nonclassical Estrogen Receptor Pathways

ERα acts either through classical (ERE‐mediated) or nonclassical (non‐ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice.

Effects of Chronic Estrogen Treatment on Modulating Age-Related Bone Loss in Female Mice

While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age‐related bone loss, we sham‐operated, ovariectomized, or ovariectomized and estrogen‐replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin–) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six‐month‐old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham‐operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen‐treated mice did not prevent age‐related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age‐related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham‐operated mice. As compared with cells from young mice, lin– cells from aged/sham‐operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age‐related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss.

TGF-β Mediates Suppression of Adipogenesis by Estradiol through Connective Tissue Growth Factor Induction

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Age-dependent renal cortical microvascular loss in female mice

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18-22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20-200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm², P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm², P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200-500 μm) decreased only in aged sham (P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.

Loss of ERE binding activity by estrogen receptor-α alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells†

Estrogen receptor (ER)‐α can signal either via estrogen response element (ERE)‐mediated pathways or via alternate pathways involving protein–protein or membrane signaling. We previously demonstrated that, as compared to wild type (WT) controls, mice expressing a mutant ER‐α lacking the ability to bind EREs (non‐classical estrogen receptor knock‐in (NERKI)) display significant impairments in the skeletal response to estrogen. To elucidate the mechanism(s) underlying these in vivo deficits, we generated U2OS cells stably expressing either WT ER‐α or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER‐α, even in the absence of E2, resulted in an increase in mRNA levels for alkaline phosphatase (AP, by 400%, P < 0.01) and a decrease in mRNA levels for insulin growth factor‐I (IGF‐I) (by 65%, P < 0.001), with no effects on collagen I (col I) or osteocalcin (OCN) mRNA levels. By contrast, stable expression of the NERKI receptor resulted in the suppression of mRNA levels for AP, col I, OCN, and IGF‐I (by 62, 89, 60, and 70%, P < 0.001). While E2 increased mRNA levels of AP, OCN, col I, and IGF‐I in ER‐α cells, E2 effects in the NERKI cells on AP and OCN mRNA levels were attenuated, with a trend for E2 to inhibit col I mRNA levels. In addition, E2 had no effects on IGF‐I mRNA levels in NERKI cells. Collectively, these findings indicate that ERE signaling plays a significant role in mediating effects of estrogen on osteoblastic differentiation markers and on IGF‐I mRNA levels. J. Cell. Biochem. 103: 896–907, 2008.

Clinical, Cellular and Molecular Phenotypes of Aging Bone

Our understanding of gerontological bone loss and osteoporosis has grown substantially in the recent past. Clinical as well as basic and translational studies have been pivotal in providing us with the pathophysiology of this condition. They have also informed us of the various cellular and molecular mechanisms underlying age related bone loss. This chapter focuses on the current concepts and paradigms of age related bone loss in humans and how various animal and cellular models have broadened our understanding in this fascinating but complex area. Changes in hormonal, neuronal and biochemical cues with age and their effect on bone have been discussed. This chapter also outlines recent studies on the relationship between bone and fat in the marrow, and the fate of the marrow mesenchymal stromal cell population which can give rise to either bone forming osteoblasts or fat-forming adipocytic cells as a function of age.

TGFβ Inducible Early Gene-1 Plays an Important Role in Mediating Estrogen Signaling in the Skeleton

TGFβ Inducible Early Gene‐1 (TIEG1) knockout (KO) mice display a sex‐specific osteopenic phenotype characterized by low bone mineral density, bone mineral content, and overall loss of bone strength in female mice. We, therefore, speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. To test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell, and biochemical level. Dual‐energy X‐ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro‐CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild‐type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter and resorptive activity as determined by serum levels of CTX‐1 were differentially regulated after estrogen treatment in TIEG1 KO mice compared with wild‐type littermates. No significant differences were detected in serum levels of P1NP between wild‐type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the sex‐specific osteopenic phenotype observed in female TIEG1 KO mice.