Regulation of chick bone growth by leptin and catecholamines

MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Regulation of chick bone growth by leptin and catecholamines L. J. Mauro,1 S. J. Wenzel, and G. M. Sindberg Department of Animal Science, Physiology and Growth Division, University of Minnesota, St. Paul 55108 bromodeoxyuridine incorporation revealed no increase in bromodeoxyuridine-positive cells in the condyles in response to leptin or NE treatments. Real-time PCR analysis showed that NE enhanced type X collagen mRNA expression, a marker of mature hypertrophic chondrocytes, with no effect on type II collagen mRNA, the matrix protein secreted by proliferating chondrocytes. Leptin treatment had no effect on the expression of either matrix protein. Treatment with agonists specific for α- or β-adrenergic receptors indicates that the activation of α-adrenergic receptors is most likely responsible for the sympathetic effect on type X collagen gene expression. These results suggest that NE and other sympathetic agonists have positive effects on bone elongation and the changes in critical genes associated with this process. These neurotransmitters may facilitate this by promoting chondrocyte maturation. These studies represent novel evidence suggesting a role of sympathetic tone in the regulation of skeletal growth in avian species.

Key words: skeleton, chondrocyte, metabolism, norepinephrine, adrenergic receptor 2010 Poultry Science 89:697–708 doi:10.3382/ps.2009-00363

INTRODUCTION

Yet, we still lack a full understanding of the identity and actions of physiological regulators important in nutritional status and how this translates into a healthy skeleton at all stages of life. There is now intriguing evidence showing that the maintenance or homeostasis of the skeleton is achieved by interconnected neural and endocrine pathways (Patel and Elefteriou, 2007) and leptin is one key endocrine factor in these pathways that links the regulation of energy-nutrient balance to skeletal homeostasis (Karsenty, 2006). Leptin was originally isolated as the product of the mouse obesity (ob) gene (Zhang et al., 1994) and is now known as an anorexigenic hormone that can modulate feeding and metabolism in animals and humans. Secreted predominantly by the adipose in mammalian species, levels of this hormone are often positively correlated with the amount of body fat and the size of adipocytes in rodents and humans (Maffei et al., 1995; Considine et al., 1996). Peripheral or central (e.g., intracerebroventricular) administration of leptin results in inhibition of food intake, decreased BW, and de-

The skeleton serves many functions for a bird: locomotion, organ support, and dynamic storage site for calcium, an essential mineral for egg laying (Karaplis, 2002). Because of this, the health of the skeleton is critical for growth and reproduction. The selection of poultry for rapid growth rate, increased muscle mass, and high egg production has resulted in poor skeletal health and an increasing prevalence for skeletal disorders, with related losses reaching hundreds of millions of dollars per year (Julian, 1998; Rath et al., 2000). Diet and nutritional status have traditionally been the most relevant factors in the management of skeletal health in avian species and consideration of these factors has helped to mitigate such losses (Edwards, 2000). ©2010 Poultry Science Association Inc. Received July 17, 2009. Accepted January 12, 2010. 1 Corresponding author: [email protected]

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ABSTRACT Leptin and the sympathetic nervous system have a unique role in linking nutritional status to skeletal metabolism in mammals. Such a regulatory mechanism has not been identified in birds but would be beneficial to signal information about energy reserves to an organ system essential for locomotion, reproduction, and survival. To explore this potential role of leptin and the sympathetic nervous system in birds, an ex vivo chick tibiotarsal model was used to test the effects of leptin and sympathetic activity on longitudinal bone growth and the expression of chondrocyte markers. Reverse transcription-PCR analysis revealed the expression of chicken leptin receptor mRNA as well as both α-adrenergic (α1A, α2A, α2B, α2C) and β adrenergic (β1, β2) receptor subtype mRNA in the whole bone. Incubation with norepinephrine (NE; 0, 10, or 100 µM for 4 d) caused a significant increase in distal condyle length as compared with vehicle-treated, contralateral tibiotarsi. In contrast, no change in condyle length was detected after leptin treatment (0 or 10 nM or 1 µM for 4 d). Analysis of cell proliferation by

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thetic activity without effect on weight or food intake, causes a significant increase in bone mass (Gordeladze and Reseland, 2003). It has also been observed that leptin can have anabolic effects when administered peripherally in vivo (Steppan et al., 2000) or directly to in vitro osteoblast cultures (Gordeladze et al., 2002), enhancing bone formation. Overall, in various states of energy restriction or abundance, leptin will direct changes in feed intake and energy metabolism as well as maintain skeletal homeostasis through peripheral or central effects. These discoveries have created a paradigm shift in the view of basic bone biology and nutrition. As such, it seems likely that this type of regulatory link between energy balance and skeletal homeostasis would be conserved across avian and mammalian species. Yet, there have been no published studies conducted to explore the potential role of leptin and its neural circuitry in the regulation of bone growth and metabolism in birds. In the studies presented here, we have used an ex vivo chick tibiotarsal model to determine the direct effects of leptin and sympathetic activity on longitudinal bone growth. We show that the tibiotarsi express the leptin receptor as well as several adrenergic receptors that could bind sympathetic neurotransmitters. The sympathetic agonist, NE, can increase the growth of the cultured tibiotarsi and enhance the expression of type X collagen, a matrix protein expressed by mature chondrocytes in developing long bone. Other sympathetic agonists show similar effects with some specificity for adrenergic receptor subtypes. To our knowledge, this is the first evidence to date suggesting that activation of adrenergic receptors may result in the modulation of skeletal growth in avian species.

MATERIALS AND METHODS Dissections and Organ Cultures Fertilized specific-pathogen-free eggs were purchased from Charles River Laboratories (Avian Products and Services, North Franklin, CT) and incubated for 10 d. Contralateral pairs of tibiotarsi were dissected from each d 10 chicken embryo and connective tissue was carefully removed. One bone of each pair was used in all experiments as an internal control treated with vehicle and the other bone was treated with the indicated hormone or agonist. This approach compensates for the inherent variability of embryonic growth rates that occurs between eggs. This variability accounts for the differences in starting bone or condyle length observed in our data. In experiments in which longitudinal measurements or bromodeoxyuridine (BrdU) incorporations were performed, the proximal condyle of each tibiotarsi was also removed. Bones from each embryo remained paired and were treated as control or treated bones throughout each experiment. Cultures were maintained for 2 to 5 d in serum-free BGJb media (Invitrogen/

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creased fat (Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995). These studies support the view that leptin acts as an adipostat that communicates the state of energy balance and energy reserves. Leptin signals nutritional status to the brain by modulating the activity of neurons within select hypothalamic nuclei (Koyama et al., 1998; Cowley et al., 2001). These neurons subsequently activate additional downstream circuitry, linking to the peripheral autonomic nervous system with innervation of peripheral tissues such as adipose and bone (Zigman and Elmquist, 2003; Chenu, 2004). In addition, leptin is able to act directly on many tissues. The overall effect of this hormone is to increase energy expenditure and decrease food intake. In avian species, less is known about the function of leptin and its neuroendocrine pathways that regulate energy balance. The studies reporting the cloning and expression of the chicken leptin gene are very controversial due to its high sequence homology with mammalian leptins (Friedman-Einat et al., 1999; Scanes, 2008; Sharp, 2008). Functional experiments indicate that both intracerebroventricular and peripheral leptin administration can reduce feed intake in chickens (Denbow et al., 2000; Taouis et al., 2001) and wild species (Lõhmus et al., 2003). There is strong evidence for the existence of the avian leptin receptor, which is structurally similar to the mammalian long-form Ob-Rb and is highly expressed in the hypothalamus as well as the lung, liver, and adipose (Ohkubo et al., 2000; Richards and Poch, 2003). Upon ligand binding, this receptor couples with and activates the JAK-STAT signaling pathway as observed for the functional isoform in mammals (Adachi et al., 2008). However, even though the expression of functional leptin receptors in the avian hypothalamus would suggest the existence of leptin-hypothalamus-peripheral neural circuitry, little research has been conducted to confirm the conservation of this regulatory loop in birds. Surprisingly, it was recently discovered that leptin, in addition to its regulation of energy expenditure and food intake, may have a unique role in linking nutritional status to bone metabolism. Studies have shown that this hormone is a potent inhibitor of bone formation when administered centrally in the hypothalamus in rodents (Ducy et al., 2000). Leptin-deficient, leptin receptor-deficient, and the fat-free mice all exhibit a higher bone mass with a 60 to 70% greater bone formation rate (Karsenty, 2001). It is believed that the sympathetic nervous system is the link between the hypothalamus and the peripheral bone tissue, responsible for leptin’s negative effects on bone mass (Takeda et al., 2002). Mice deficient in dopamine β-hydroxylase, an enzyme necessary to produce epinephrine (EPI)norepinephrine (NE), have little to no sympathetic activity-tone, exhibit a high bone mass phenotype, and are resistant to the antiosteogenic effects of leptin. Longterm treatment of normal mice with a β-adrenergic antagonist known as propranolol, which blocks sympa-

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Measurements To determine effects of treatments on longitudinal bone growth, digital images of the distal condyle and adjacent shaft of each bone were taken. Using image measurement software (Image ProJ, National Institutes of Health, Bethesda, MD), condyle length was measured from the end of the distal condyle to the beginning of the shaft-bony collar. Units were corrected for magnification and expressed as millimeters. Preliminary experiments using Alizarin Red (bone) and Alcian Blue (cartilage) staining of cultured bones indicated that this junction could be consistently distinguished by the outgrowth or bulge of cartilaginous tissue formed at this junction during the culture period. Thus, in subsequent experiments, imaging and measurement of fresh unstained bones were performed, avoiding the variability due to dye and alcohol treatments.

BrdU Incorporation Preliminary experiments with tibiotarsi lacking the proximal condyle indicated that the cartilaginous tissue of the distal condyle was permeable to BrdU and that consistent peak numbers of BrdU-positive cells were observed after an 8-h incubation. In subsequent experiments, tibiotarsi pairs were treated for 72 h with sympathetic agonists or hormones as indicated, then 10 µM BrdU was added for 8 h. A total of 3 pairs were collected per treatment group. At the end of this incubation, individual bones were rinsed for 5 min in 1× PBS twice then transferred to 10% neutral-buffered formalin for overnight incubation at 4°C. Decalcification of samples was performed by incubation in 10% EDTA (pH 6.0) overnight followed by water rinses and storage in 70% alcohol. Bones were then processed as contralateral pairs, paraffin-embedded, and cut into full-length 4-µm sections. From each bone, 6 sections were collected in 3 sets of 2 adjacent sections with an approximately 80-µm interval between sets. This provided a robust sampling of the cartilaginous condyle for subsequent immunohistochemical analysis of BrdU incorporation.

Immunohistochemistry and Slide Analysis The BrdU In Situ Detection Kit (BD Biosciences, San Diego, CA; Dover and Patel, 1994) was used to detect BrdU-positive cells in the tibiotarsi. The manufacturer’s protocol was used with modifications. Slides were deparaffinized in xylene, washed in ethyl alcohol, and rinsed in distilled water and 1× PBS with 0.05% Tween 20. Hydrogen peroxide treatment (3%, 10 min., room temperature) was performed followed by antigen retrieval in 10 mM of heated citrate (pH 6.0) for 20 min. Primary and secondary antibody incubations as well as the 3,3’-diaminobenzidine substrate reaction were conducted as outlined in the kit manual. All slides were counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped using Permount (Thermo Fisher Scientific, Pittsburgh, PA). The image analysis software, MetaMorph (v.7.1.0, Molecular Dynamics, Sunnyvale, CA), was used to determine the number of BrdU-positive cells in sections from control and treated bone pairs. For each adjacent section within a set, the total count of positive cells within the entire bone section was taken. The average number of cells within the 2 adjacent sections was determined and these averages for each set were added to calculate the approximate number of BrdU-positive cells within each bone. The following equation was used to compare control versus treated tibiotarsi and calculate normalized units of change: {([BrdU cells]treated − [BrdU cells]control)/ [BrdU cells]control} + 1.00.

RNA Extractions Individual bones were collected on the days indicated, flash frozen in liquid nitrogen, and stored at −80°C. In addition, adult chicken brain and liver tissue were also collected from killed birds as positive control samples for PCR analysis described below. The RNA extractions were carried out using the RNeasy Kit (Qiagen, Valencia, CA; Schiller et al., 2009) with minor modifications. To enhance extraction efficiency from bone tissue, individual bones were ground to a fine powder in liquid nitrogen using a mortar and pestle. The remainder of the extraction followed the manufacturer’s protocol for isolation of RNA from difficult tissues (appendix C; kit manual). Chicken heart RNA was purchased from Zyagen (San Diego, CA) and turkey heart RNA was provided by Kent Reed (University of Minnesota); both served as additional control tissues for adrenergic receptor expression analyses. All RNA samples were treated with DNase I using standard protocols (Turbo DNA-Free Kit; Ambion, Austin, TX; Schiller et al., 2009).

PCR Analysis Conventional reverse transcription-PCR (RT-PCR) analysis was performed to verify the expression of the

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Gibco, Carlsbad, CA) plus supplements (1% penicillinstreptomycin, 1 mM β-glycerol phosphate and 50 µg/ mL of ascorbic acid; Invitrogen/Gibco) in an incubator at 37°C and 5% CO2. Drugs and hormones used included transforming growth factor-β (R&D Systems, Minneapolis, MN), chicken leptin (National Hormone and Pituitary Program, Torrence, CA), NE (Sigma, St. Louis, MO), epinephrine hydrochloride (Sigma), phenylephrine (PHE; Sigma), and isoproterenol (ISO) hydrochloride (Sigma). These substances were prepared in Nanopure Water (Milli-Q Continental Water Systems, Billerica, MA) or according to the manufacturer’s protocols just before media changes and were added to cultures every 2 d.

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Statistical Analysis For total bone and condyle length, an unpaired Student’s t-test was performed with significance at P < 0.05. For BrdU incorporation data and Q-PCR analyses, a nonparametric test known as Wilcoxon signed rank test was performed in which a significant difference from the assigned value of 1.0 (e.g., no change) was set for P-values <0.05.

RESULTS Bone Culture Validation Initial experiments were conducted to establish the ex vivo culture. Contralateral pairs of the long leg bone, the tibiotarsus, were isolated from chick embryos at d 10 of incubation (Figure 1A). After dissection, the bones were placed in separate wells of culture media and examined to ensure that the proximal and distal condyles were intact and the bone shaft (diaphysis) was undamaged (Figure 1B). The presence of a primary ossification site and a bony collar within the transparent diaphysis was often observed. Measurements of these bones taken on d 0 and 5 of culture showed a significant increase in total length, verifying that these bones will grow in these culture conditions (Figure 1C). In addition, treatment of these bones with transforming growth factor-β1 (TGF-β1; 10 ng/mL) resulted in a significant decrease in bone growth by d 5 of culture (Figure 1D). Transforming growth factor-β1 is a known inhibitor of chondrocyte proliferation and has been shown to reduce the length of cultured metatarsal bones from mouse embryos (Dieudonné et al., 1994) and tibiotarsal bones from chick embryos (Crochiere et al., 2008). The observed effects of TGF-β1 on the tibiotarsus support these studies and provide further evidence that these

Table 1. Primers and amplicon size for chicken genes analyzed by reverse transcription-PCR and quantitative real-time PCR Gene name Leptin receptor1

Accession no.

Forward (For) and reverse (Rev) primers

NM_204323

For 5′-CTTCTGGAACCGGAAATAGTG-3′ Rev 5′-CTTGCTGTTTCTCCAGAGGTC-3′ For 5′-AAATGTCCTGAGAGCGCAGT-3′ Rev 5′-AGGAATCGCACTTTTTGTCG-3′ For 5′-CCTCTGAGCACCAGGAGAAC-3′ Rev 5′-ACGGGGTTCAAGGAGCTATT-3′ For 5′-AGGATGCAGTTGGGAGAGAA-3′ Rev 5′-CACAGCCAGCACAAAGGTAA-3′ For 5′-TCGCTGGACGTGCTTTGCGT-3′ Rev 5′-CCCGGTTGGTGACAAAGTCGCA-3′ For 5′-AACAAGGAGCAGGACCAGAA-3′ Rev 5′-TGCCGTTGCTGTTTGAGTAG-3′ For 5′-AGGCAGTGCTGTCATTGATCT-3′ Rev 5′-GCCCAGTTAAAATGTCCTGAA-3′ For 5′-TTACTGGATTGACCCGAACC-3′ Rev 5′-GCCGTAGCTGAAGTGGAAAC-3′ For 5′-GGAGAAACCAGCCAAGTATGA-3′ Rev 5′-AAACAAGCTTGACGAAATGGT-3′

receptor1

XM_425762

α2A-Adrenergic receptor1

XM_426537

receptor1

XM_426355

α1A-Adrenergic

α2C-Adrenergic

β1-Adrenergic receptor1 β2-Adrenergic

receptor1

Type X collagen2 Type II

collagen2

Glyceraldehyde 3-phosphate dehydrogenase2 1Analyzed 2Analyzed

by reverse transcription-PCR. by quantitative real-time PCR.

M14379 XM_425195 M13496 AY046949 V00407

Amplicon size (nt) 206 229 337 321 262 340 189 228 188

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genes encoding the chicken leptin receptor as well as the known chicken adrenergic receptors. Complementary DNA from bone, brain, heart, and liver tissues was synthesized from isolated RNA samples using the Retroscript Kit (Ambion; Schiller et al., 2009) and including a no reverse transcription control. Multiple pairs of primers were designed for each gene using Primer 3 software (http://frodo.wi.mit.edu/primer3/) and reaction conditions were optimized for the production and size of the expected amplicon. Final primers (Table 1) were used under standard PCR methodology including both no DNA and no reverse transcription controls. Amplicons were resolved on 4% NuSieve gels (Lonza Rockland Inc., Rockland, ME). Quantitative real-time PCR (Q-PCR) analysis was performed to monitor changes in the expression of type II and type X collagen genes during treatments. Complementary DNA from individual bone RNA samples was synthesized using the TaqMan reverse transcriptase reagents (ABI, Foster City, CA). Primers were specifically designed for Q-PCR using Primer 3 software and were tested using RT-PCR to verify the production and size of the amplicons (Table 1). The Q-PCR reactions were conducted with SYBR Green dye for amplicon detection using manufacturer’s protocol for the Brilliant SYBR Green QPCR Master Mix Kit (Stratagene, La Jolla, CA; Schiller et al., 2009). Dissociation curves for each primer set and gel electrophoresis analysis of Q-PCR reactions were used to verify a single amplicon. When possible, multiple lots of cDNA were tested to ensure reproducibility. The ΔCt scores for chicken type II and type X collagen transcripts in each sample were normalized using ΔCt data for chicken glyceraldehyde 3-phosphate dehydrogenase and were expressed as the fold change of control versus treated using the following equation: fold change = 2−ΔΔCt.

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bones are viable and capable of responding to modulators of bone growth in this culture system.

Expression of the Leptin and Adrenergic Receptors To determine if the cells of the tibiotarsus could potentially respond to leptin or catecholaminergic neurotransmitters, the expression of the leptin receptor and of select adrenergic receptors was examined in RNA collected from tibiotarsi. The RT-PCR analysis indicated that the leptin receptor transcript is expressed in both freshly dissected as well as d 5 cultured bones (Figure 2A). As expected, this transcript is also expressed in the brain and the liver of adult laying hens. Analysis of adrenergic receptor expression showed that specific amplicons could be detected for all the α-adrenergic receptors examined (α1A, α2A, α2B, α2C) as well as the β2-adrenergic receptor (Figure 2B). Because original primer pairs failed to detect a transcript for the β1adrenergic receptor, additional pairs were designed and RT-PCR was conducted with chicken heart RNA as a control because this tissue has been shown to express this gene (Sommer et al., 2005). Amplification from control chick heart RNA gave a strong band, whereas a

relatively weak signal was detected from the chick bone (Figure 2C). Therefore, β1-adrenergic receptor subtype may also be expressed in d 10 embryonic whole tibiotarsi.

Effects of Leptin and NE on Longitudinal Bone Growth Distal condyle length was used as an estimate of longitudinal bone growth (Figure 3A). This measurement was used in place of total bone length to reduce variability due to normal bone curvature during growth in ex vivo cultures. Treatment of tibiotarsi with leptin for 4 d resulted in no change in condyle length in the bones cultured with 10 nM or 1 µM leptin as compared with its untreated contralateral pair (Figure 3B;10 nM: control, 4.03 ± 0.03 vs. treated, 4.22 ± 0.05; 1 µM: control, 3.93 ± 0.07 vs. treated 4.22 ± 0.08). In contrast, incubation with the sympathetic agonist, NE, resulted in a significant increase in condyle length at both the 10 µM and the 100 µM concentrations (Figure 3C; 10 µM: control, 4.50 ± 0.13 vs. treated, 5.01 ± 0.14; 100 µM: control, 3.92 ± 0.08 vs. treated, 4.36 ± 0.05). Treatment of bones with the control bone modulator, TGF-β1, caused a significant reduction in con-

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Figure 1. Ex vivo cultures of embryonic chick tibiotarsi. A. Schematic of avian leg bones with location of tibiotarsus indicated by arrow. Bones were dissected from d 10 chick embryos and placed in a culture well as shown in panel B and described in Materials and Methods. The proximal and distal condyles are visible (arrowheads) and a bony collar is evident in the central region of the diaphysis (arrow). C. Total bone lengths (mm) measured on d 0 and 5 of culture indicate viable tissue. D. Transforming growth factor β1 (TGF-β1) inhibits this normal growth. Bars indicate mean ± SE, n = 4 to 6 bones per group. Different letters above bars indicate significant differences between means with P-values as c ≤ 0.001 and d ≤ 0.0001. Color version available in the online PDF.

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dyle length (Figure 3D). These results indicate that the catecholamine NE can enhance longitudinal growth of chick embryonic bones.

Effects of Leptin and NE on Chondrocyte Proliferation

Effects of Leptin, NE, and Other Sympathetic Agonists on Matrix Expression Proliferating chondrocytes and articular chondrocytes synthesize a matrix primarily composed of type II collagen (Karaplis, 2002). Once arrested in the cell cycle, these cells continue to differentiate to become mature hypertrophic chondrocytes that express type X collagen and mineralize the cartilage matrix. This progression from proliferating to hypertrophic chondrocytes drives longitudinal bone growth. The expression of both type II and type X collagen in control and treated tibiotarsi pairs was examined to test if leptin or NE was modulating the expression of these genes. Preliminary experiments were conducted to determine the optimal time for incubation and RNA collection for each treatment. Bone pairs were treated with leptin (10 nM or 1 µM) for 4 d, collected, and RNA was extracted for quantitative real-time PCR analysis. Those bones treated with leptin showed no significant change in either type II or type X collagen mRNA (Figure 5A). In growth studies, NE was effective at the lower 10µM dose, which is the concentration often used in in vitro bone studies (Figure 3). Also, preliminary experiments indicated potential changes in expression at both d 2 and 4 of culturing. Therefore, subsequent experiments were conducted using 10 µM NE treatment of tibiotarsi pairs and RNA collections made at d 2 and 4. On d 2 of treatment, a 2.7-fold increase in type X expression was observed in treated bones (2.7 ± 0.46; P = 0.03), whereas no change was seen in type II expression (Figure 5B). Extending treatment to 4 d had no effect on the expression of either collagen gene. Treatment of tibiotarsi pairs with the negative control, TGF-β1, resulted in a significant reduction (30 to 50%) in type II expression [Figure 5C; d 2: 0.69 ± 0.06 (P = 0.02); D4:

Figure 2. Expression of the leptin receptor and α- and β-adrenergic receptors in the tibiotarsus. Bones (fresh or cultured for 5 d) as well as brain and liver tissue (laying hens) were collected and processed in parallel for reverse transcription-PCR. A. Gel of PCR analysis for the leptin receptor from cultured (Cult) and fresh (Fr) bones as well as brain (Br) and liver (Lvr) shows that the tibiotarsi express the leptin receptor. B. Analysis for α-adrenergic (α1A, α2A, α2C) and β2-adrenergic receptors from cultured bones. Representative negative controls without cDNA [(−)d] and with an aliquot of cDNA reaction lacking reverse transcriptase [(−)rt] are shown. C. Analysis for β1 subtype from chick heart (H) and tibiotarsus (Bo) RNA. Samples in lanes 1 = (−)rt, H; 2 = H, 4 µL of cDNA; 3 = H, 2 µL of cDNA; 4 = (−)rt, Bo; 5 = Bo, 4 µL of cDNA; 6 = Bo, 2 µL of cDNA; 7 = (−)d. Corresponding base pairs for molecular weight markers are indicated for panels A to C.

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Longitudinal bone growth is due to tightly regulated changes in chondrocyte proliferation and maturation. To determine if leptin or NE can modulate chondrocyte proliferation, control and treated tibiotarsi were incubated with 10 µM BrdU and processed for immunocytochemical detection of proliferating, BrdU-positive cells. The protocol for BrdU incorporation was effective in labeling and detecting proliferating chondrocytes throughout the distal condyle and within the adjoining diaphysis (Figure 4A). Analysis of total BrdU-positive cells revealed no significant difference between control tibiotarsi and the contralateral pair treated with leptin or NE (Figure 4B).

0.54 ± 0.04 (P = 0.004)]. Little effect on type X collagen expression was observed after TGF-β1 treatment. To further explore the modulatory role of sympathetic activity on chondrocyte gene expression, we examined the effect of other adrenergic agonists. The α subtypes of the adrenergic receptor expressed by heart and muscle tissue, the extensively studied targets of sympathetic activation, are often more sensitive to the catecholaminergic neurotransmitter, EPI (Westfall and Westfall, 2006). The synthetic agonists, PHE and ISO, are often more effective in activating the α1 and β(1, 2, 3) subtypes, respectively. None of these receptor subtypes are well characterized in skeletal tissue and this is especially the case in avian species. We tested these 3 agonists and measured changes in type II and X collagen mRNA expression after 2 d of culture because NE was most effective at this time point. Treatment of

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tibiotarsi pairs with vehicle or 10 µM EPI resulted in a ~2-fold increase in type X mRNA (2.19 ± 0.31; P = 0.001) with no significant effect on type II (Figure 6; left graph). Bones treated with 10 µM PHE also showed a significant increase in type X expression (2.30 ± 0.26; P = 0.001) with no change in type II collagen (Figure 6; middle graph). In contrast, ISO treatment had no effect on either type II or type X collagen mRNA expression (Figure 6; right graph).

DISCUSSION

bone (Ducy et al., 2000; Takeda et al., 2002). To our knowledge, there are no published reports on the putative role of this regulatory pathway in the avian skeleton. Our studies presented here examined the effects of leptin and sympathetic neurotransmitters on chick bone growth using an ex vivo tibiotarsus model. We have observed that the activation of sympathetic input, through adrenergic agonist stimulation, can increase longitudinal bone growth and enhance type X collagen mRNA expression. In contrast, the leptin hormone appears to have no direct effect on these parameters of bone development using this model. Analysis of whole tibiotarsi revealed the expression of mRNA encoding several adrenergic receptors as well as the leptin receptor. Both fresh and cultured tibiotarsi express the genes encoding the known chicken α (α1A, α2A, α2C)-receptors, the β1- and β2-adrenergic receptor, and the leptin receptor, although the specific cell

Figure 3. Effect of chicken leptin and norepinephrine on longitudinal growth of the tibiotarsus. A. Photomicrograph showing method of condyle measurements. Digital images of bone were recorded and the distance from the junction of the condyle (epiphysis) and the bone shaft (diaphysis; left plain line on photo) to the end of the distal condyle (right plain line) was measured as described. Line with arrowheads indicates the length of the distal condyle as measured in these experiments. B, C, and D. Graphs depicting the change in condyle length after treatment of tibiotarsi with leptin (B; 10 nM or 1 µM), norepinephrine (C; 10 or 100 µM), or transforming growth factor-β1 (TGF-β1) (D; 10 ng/mL). Co = control; Trtd = treated. Contralateral bone pairs were treated with vehicle (white bar) or the indicated concentration of hormone-neurotransmitter (black bar) for 5 d. Bars indicate mean condyle length ± SE, average n = 10 to 18. Different letters above bars indicate significant differences between means with P-values as b ≤ 0.05 and c ≤ 0.001. Color version available in the online PDF.

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Leptin can influence the mammalian skeleton via indirect or direct modes of action, resulting in either positive or negative effects on bone growth and metabolism. The central or indirect actions of this adipokine are mediated through hypothalamic neurons followed by downstream sympathetic activation of innervated

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type expressing these receptors was not determined. For adrenergic receptors, this is similar to what has been observed in other species. Studies have documented the expression of these receptor subtypes, except β1, in human and rodent osteoblast and osteoclast primary cells and cell lines (Togari et al., 1997; Takeda et al., 2002; Togari, 2002; Nishiura and Abe, 2007). The expression of the β2-adrenergic receptor in murine-cultured growth plate chondrocytes has been shown with no detectable β1 or β3 subtypes (Lai and Mitchell, 2008). The difference in our observed expression of the β1 subtype may be due to species differences (e.g., avian vs. mammalian) as well as source of bone RNA (e.g., whole bone tissue vs. isolated cells-cell lines). As for leptin receptor expression, mammalian chondrocytes express the long form of the Ob-Rb leptin receptor (Figenschau et al., 2001; Dumond et al., 2003; Ben-Eliezer et al., 2007). No published reports have addressed the expression of the adrenergic or leptin receptors in avian skeletal tissue. Incubation of the d 10 chick tibiotarsi with NE, the neurotransmitter that can activate both receptor subtypes, resulted in enhanced longitudinal growth and increased type X collagen mRNA expression with no change observed in type II collagen mRNA. These adrenergic effects on gene expression were substantiated by the similar enhancement of this gene after treatment with both EPI and PHE, an α-receptor agonist. The stimulation of β-adrenergic receptors using ISO, a β-agonist shown to be a very potent modulator of bone formation in mammalian species, had no effect in our studies. Such results suggest that adrenergic stimulation can promote bone elongation in the chick skeleton and it may be the activation of the α-adrenergic receptors in these tibiotarsi that is responsible for the change in type X collagen expression. The reason for the supposed species differences cannot be explained. It is difficult to extrapolate from in vivo systemic ad-

ministration of such β-agonists, which is most prevalent in the mammalian studies, to our ex vivo bone culture. In addition, the in vitro rodent studies have not been conducted with this type (e.g., endochondral long bone vs. intramembraneous bone) and age (e.g., embryonic vs. neonate or adult) of skeletal tissue. It should also be noted that the balance of the sympathetic regulation of bone metabolism in the adult mammalian skeleton is not totally clear because osteoblasts and osteoclasts express both α- and β-adrenergic receptors and selective activation of these can result in anabolic or catabolic effects depending on the context (Togari, 2002). So, like leptin, negative or positive effects of adrenergic stimulation can be observed, illustrating the need for further studies to understand its role in the vertebrate skeleton. The long bones of vertebrates start as a cartilage template during embryonic development and undergo endochondral ossification to form the final skeletal element. It is the progressive maturation of the chondrocytes of the developing long bone that drives linear growth, with proliferating chondrocytes secreting predominantly type II collagen and the prehypertrophic to hypertrophic mature cells expressing type X collagen (Shum and Nuckolls, 2002). In general, bone elongation is thought to require enhanced chondrocyte proliferation and often is accompanied by suppressed maturation of the chondrocyte population. Indeed, in one of the few studies on chondrocytes and adrenergic regulation, treatment of cultured mouse growth plate chondrocytes with ISO enhanced proliferation but inhibited type X collagen expression and maturation (Lai and Mitchell, 2008). We observed in our tibiotarsi cultures a significant increase in condyle length after NE treatment as well as enhanced type X collagen expression but no change in the number of BrdU-positive cells. This suggests that this adrenergic agonist is potentially

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Figure 4. Chondrocyte proliferation after leptin and norepinephrine treatment. A. Photomicrograph of a section showing bromodeoxyuridine (BrdU)-positive chondrocytes (brown staining; arrowheads) within the distal condyle. After 72 h of treatments, tibiotarsi were incubated with 10 µM BrdU for 8 h and bones were processed as indicated. Bar = 22.5 µm. B. Graph depicting change in numbers of proliferating chondrocytes in control versus treated bones during treatment with leptin (Lep; 10 nM or 1 µM) or norepinephrine (NE). Bars indicate mean fold change ± SE, average n = 3 bones per group. No significant differences in normalized units of change were detected for any of the treatments. Color version available in the online PDF.

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promoting linear growth via chondrocyte maturation without affecting chondrocyte proliferation. As incongruous as this may appear, the model systems used, the complexity of the hormonal pathways being examined, as well as variable, localized effects within the cartilage template can produce such results. For example, in the developing chick wing, overactivation of phospholipase C signaling, a pathway important in chondrocyte

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maturation, results in increased length of limb bones along with regions of premature, accelerated chondrocyte maturation, which may or may not be associated with changes in the proliferative zone (Taschner et al., 2008). In studies with chick tibiotarsi, cartilage lengthening, expansion of the proliferative zone, and enhanced expression of type X collagen are all observed in the absence of the perichondrium (Long and Linsenmayer,

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Figure 5. Effect of chicken leptin and the neurotransmitter, norepinephrine, on chondrocyte expression of type II and type X collagen mRNA. Bones were collected after treatments and quantitative real-time PCR was performed. Data are calculated as fold change in the relative amount of each gene transcript expressed by control bones as compared with treated bones. A. Effect of leptin treatment (10 nM or 1 µM) for 4 d. B. Effect of norepinephrine treatment (10 µM) for 2 and 4 d. C. Effect of transforming growth factor-β1 (10 ng/mL) for 2 and 4 d. Bars indicate mean fold change ± SE, average n = 6 to 9 bones per group. Asterisk above bar indicates significant difference from fold change of 1 at P < 0.05 as determined by Wilcoxon signed rank test.

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Figure 6. Effect of additional adrenergic agonists on the gene expression of type II and type X collagen mRNA in the tibiotarsus. Bones were collected after treatments and quantitative real-time PCR was performed. Data are calculated as fold change in the relative amount of each collagen gene transcript expressed by control bones as compared with treated bones. Effect of 10 µM epinephrine (EPI), phenylephrine (PHE), or isoproterenol (ISO) treatment for 2 d on expression of type II and type X collagen mRNA is indicated on each graph. Bars indicate mean fold change ± SE for each comparison within each collagen gene. An average n = 9 to 13 bones per group was used. Asterisk above bar indicates significant difference from fold change of 1 at P < 0.0001 as determined by Wilcoxon signed rank test.

pothalamic neurons-sympathetic innervation pathway is active in ovo and does regulate skeletal development and growth at this stage. This would be similar to the accepted leptin regulatory pathway thought to exist in mammals, although most such studies point to leptin as a potent inhibitor of bone formation (Ducy et al., 2000; Takeda et al., 2002). The fact that we observed enhancement of both longitudinal growth and potential chondrocyte maturation does not necessarily conflict with its proposed inhibitory role in mammals because leptin treatment can correct short stature in leptin-deficient mice by increasing femoral bone length (Steppan et al., 2000; Iwaniec et al., 2007). It would be presumed that this is via leptin’s central actions with downstream sympathetic activation of the bone. Our research raises the possibility that sympathetic tone is a regulator of linear skeletal growth in the chick. In our tibiotarsal explant model, NE has a positive direct effect on longitudinal growth and adrenergic agonists are able to modulate the expression of the type X collagen gene, which encodes a matrix protein secreted by mature chondrocytes. Future studies should address the identification of specific cell types expressing the leptin and adrenergic receptors, the localization of sympathetic innervation within the developing bone, and the effect of in ovo manipulation of such neural tone on skeletal development. Such additional research would support the concept that avian species in general have the circuitry to allow for this type of regulation of skeletal growth during development. Also, it would be interesting to explore the role of both leptin and sympathetic tone on skeletal metabolism in the adult bird.

ACKNOWLEDGMENTS We thank Marion Zillhardt, Audrey Mayer, Ngozika Okoye, and Courtney Blohm (University of Minnesota) for excellent technical assistance. Kent Reed (University of Minnesota) generously donated turkey heart RNA for the initial verification of PCR primer sets used for amplification of the turkey and chicken β1-adrenergic receptor genes. Cathy Carlson and Ann Undersander (University of Minnesota) provided invaluable advice

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1998). Further studies in avian models are obviously necessary to verify the adrenergic effects on chondrocyte maturation and clarify the mechanisms involved. The concept that leptin or sympathetic activity can modulate bone elongation would require the presence of these factors during embryonic development and the period of posthatch or neonatal growth of the skeleton. Sympathetic innervation of the mammalian skeleton is supported by the presence of nerve fibers expressing catecholamine-synthesizing enzymes in neonatal and adult bone (Hill and Elde, 1991; Mach et al., 2002) as well as the modulation of bone growth and resorption by chemical or surgical sympathectomy (Singh et al., 1981; Sherman and Chole, 1996). Sensory neuronal innervation of the developing chick limb bud has been observed by stage 30 (~6.5 d of incubation), but little to no chemical or functional characterization of these neurons within the bone tissue has been conducted (Wang and Scott, 2000). The limited research in the chick suggests that the d 10 tibiotarsi in our model may have a functional neural network as well as bone and cartilage cells capable of responding to sympathetic regulation during development. As for leptin, leptin receptor and protein are expressed in the embryo, fetus, and placenta of many mammals and leptin levels are correlated with linear growth rates and stature of the fetus and neonate (Gat-Yablonski and Phillip, 2008). The embryo and the yolk sac of a chicken egg express the leptin receptor as early as d 5 of incubation as well as at d 12 and 17 (Ashwell et al., 1999; Hu et al., 2008). But, specific expression in the musculoskeletal system of any avian species has not been reported. The results of our leptin treatment experiments suggest that leptin, or a similar ligand that activates the leptin receptor, does not modulate longitudinal growth or the expression of chondrocyte markers, at least within the confines of our ex vivo model. This does not necessarily mean that such regulation of the skeleton does not exist or function in avian species but rather that, at this developmental stage, it does not appear to be acting directly on cells within this skeletal element. Because we did observe an effect by mimicking direct sympathetic activation, it is possible that the leptin-hy-

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on bone histopreparation and immunostaining. This research was supported by funds from the Minnesota Agricultural Experiment Station (to LJM: MIN-16-016).

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