Functional adaptation in female rats: the role of estrogen signaling.

Sex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERβ), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females.We used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17β-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERβ, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced.Estrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats

Role of Calcitonin Gene-Related Peptide in Functional Adaptation of the Skeleton

<div><p>Peptidergic sensory nerve fibers innervating bone and periosteum are rich in calcitonin gene-related peptide (CGRP), an osteoanabolic neurotransmitter. There are two CGRP isoforms, CGRPα and CGRPβ. Sensory fibers are a potential means by which the nervous system may detect and respond to loading events within the skeleton. However, the functional role of the nervous system in the response of bone to mechanical loading is unclear. We used the ulna end-loading model to induce an adaptive modeling response in CGRPα and CGRPβ knockout mouse lines and their respective wildtype controls. For each knockout mouse line, groups of mice were treated with cyclic loading or sham-loading of the right ulna. A third group of mice received brachial plexus anesthesia (BPA) of the loaded limb before mechanical loading. Fluorochrome labels were administered at the time of loading and 7 days later. Ten days after loading, bone responses were quantified morphometrically. We hypothesized that CGRP signaling is required for normal mechanosensing and associated load-induced bone formation. We found that mechanically-induced activation of periosteal mineralizing surface in mice and associated blocking with BPA were eliminated by knockout of CGRPα signaling. This effect was not evident in CGRPβ knockout mice. We also found that mineral apposition responses to mechanical loading and associated BPA blocking were retained with CGRPα deletion. We conclude that activation of periosteal mineralizing surfaces in response to mechanical loading of bone is CGRPα-dependent <i>in</i><i>vivo</i>. This suggests that release of CGRP from sensory peptidergic fibers in periosteum and bone has a functional role in load-induced bone formation.</p></div

Schematic diagram of the rat ulna loading model.

<p>The antebrachium was placed horizontally in loading cups attached to a materials testing machine. The medio-lateral diaphyseal curvature of the rat ulna is accentuated through axial compression, most of which is translated into a bending moment, which is greatest at ∼60% of the total bone length measured from the proximal end of the ulna <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043215#pone.0043215-Kotha1" target="_blank">[30]</a>. Reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043215#pone.0043215-Sample2" target="_blank">[5]</a> with permission from John Wiley & Sons.</p

Estrogen receptor expression in brachial intumescence (C₆-T₂) dorsal root ganglia gene.

<p><b>Note</b>: Data represent mean ± standard deviation.</p>*<p><i>p</i><0.05 versus the estrogen treated group.</p>#<p>Significantly different from internal control (trigeminal ganglion) (<i>p</i><0.05).</p>a<p>Significantly different from a hypothesized mean of zero (<i>p</i><0.05). DRG = dorsal root ganglia; ERα - estrogen receptor alpha; ERβ = estrogen receptor beta; GPER – G-protein couple estrogen receptor.</p

Endosteal bone formation after mechanical loading of the right ulna in ovariectomized female rats.

<p><b>Note</b>: Data represent mean ± standard deviation.</p>*<p><i>p</i><0.05;</p>**<p><i>p</i><0.01;</p>***<p><i>p</i><0.001 versus the estrogen treated group.</p>a<p><i>p</i><0.05 versus the associated Load group that did not receive brachial plexus anesthesia (BPA) before loading. Effect size comparing Load and BPA+Load groups for each respective treatment. En.MS/BS = endosteal mineralizing surface; En.MAR = endosteal mineral apposition rate; En.BFR/BS = endosteal bone formation rate. Relative (R-L) values were also calculated – rPs.MS/BS, rPs.MAR, and rPs.BFR/BS.</p

Oligonucleotide primers for quantitative real-time reverse-transcriptase-polymerase chain reaction.

<p><b>Note</b>: ERα and -β – estrogen receptors alpha and beta; GPER – G-protein coupled estrogen receptor; CGRPα – calcitonin gene-related peptide alpha; TRPV1 and −4 – transient receptor potential vanilloid-1 and −4; TRPA1 – transient receptor potential ankyrin-1; PGP9.5 – protein gene product 9.5.</p

Labeled ulna bone formation was suppressed with estrogen treatment.

<p>Treatment with estrogen after ovariectomy (OVX) decreased load-induced labeled bone formation. (<b>A</b>) Rats treated with either Placebo or G-1, a GPER-specific agonist, had increased periosteal bone formation after right ulna loading, when compared to rats treated with estrogen. (<b>B</b>) Estrogen treatment in rats that underwent brachial plexus anesthesia (BPA) before loading of the right ulna also resulted in a decreased amount of periosteal bone formation, compared to placebo and G-1 treated rats. White arrows indicate periosteal labeled new bone formation. Bar = 250 µm. Cr, cranial; Cd, caudal; Med, medial; Lat, lateral. Estrogen group, n = 7; Placebo group n = 7; G-1 group, n = 6, Estrogen+BPA group, n = 8; Placebo+BPA group, n = 7; G-1+BPA group, n = 6.</p

Adaptive periosteal bone responses in CGRPα wildtype and knockout mice are mainly influenced by changes in mineral apposition rate.

<p>Cyclic loading of the right ulna in CGRPα knockout mice, but not wild-type mice, induced significant changes in relative periosteal mineral apposition rate (Ps.rMAR). Blocking of periosteal relative mineral apposition rate (Ps.rMAR) and periosteal relative bone formation rate (Ps.rBFR/BS) by brachial plexus anesthesia (BPA) was also found in CGRPα knockout mice, but not wildtype mice. Sham – sham loaded group, Load – loaded group, Block + Load – BPA treatment before loading. n = 11–14 mice/group.</p

Schematic diagram of the ulna loading model.

<p>The antebrachium was placed horizontally in loading cups attached to a materials testing machine. The medio-lateral diaphyseal curvature of the ulna is accentuated through axial compression, most of which is translated into a bending moment. Reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113959#pone.0113959-Sample2" target="_blank">[18]</a> with permission from John Wiley & Sons.</p