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THE ANATOMICAL RECORD 290:155–167 (2007)

Parathyroid Hormone/Parathyroid Hormone-Related Peptide Modulates Growth of Avian Sternal Cartilage via Chondrocytic Proliferation ERIK KERN HARRINGTON,1 GAVIN W. RODDY,1 RANDY WEST,1 1,2 AND KATHY K.H. SVOBODA * 1 Biomedical Sciences, Texas A&M University System, Baylor College of Dentistry, Dallas, Texas 2 Department of Ophthalmology, Southwestern Medical Center, Dallas, Texas

ABSTRACT Parathyroid hormone (PTH; 107 to 1015 M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1–34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTHtreated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components. Anat Rec, 290:155–167, 2007. Ó 2007 Wiley-Liss, Inc.

Key words: hyaline cartilage; apoptosis; parathyroid hormone; parathyroid hormone-related peptide; chondrogenesis; PTH/PTHrP common receptor (PTH1R); type X collagen

Previously, we reported that 100 nM parathyroid hormone (PTH; 1–34) significantly increased the longitudinal growth of avian sterna in an 8-day, serum-free, whole organ culture (Harrington et al., 2004). In this study, we asked how did the increase in longitudinal growth occur. Tissue growth can be measured in terms of increases or decreases in total cell number (which balances cell proliferation with apoptosis), cell size, and/or extracellular matrix (ECM) deposition. Properties of the extracellular matrix include protein content and the hydrophilic or hydrophobic nature of those proteins. Accordingly, diffusion of water into or out of the tissue can also affect cartilage size. In addition, the sternal organ has Ó 2007 WILEY-LISS, INC.

Grant sponsor: The Baylor Oral Health Foundation, NIH National Institute of Dental and Craniofacial Research F30DE05743. *Correspondence to: Kathy K.H. Svoboda, Department of Biomedical Science, Texas A&M Health Science Center, Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, TX 75246. Fax: 214-828-8951. E-mail: [email protected] Received 18 August 2006; Accepted 31 October 2006 DOI 10.1002/ar.a.20416 Published online in Wiley InterScience (www.interscience.wiley.com).

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three unique regions of differentiation (cephalic-hypertrophic, middle-prehypertrophic, and caudal-proliferative) and consideration for differences in growth of each region should be analyzed. Chondrogenic growth in the embryonic chick is directed in part by a G-protein-coupled receptor (GPCR) signaling cascades. PTH is a GPCR signaling protein, which contributes to this hyaline cartilage development. PTH, an 84 amino acid protein, classically regulates calcium and phosphate homeostasis primarily through actions on specific receptors in kidney and bone (Juppner et al., 1991; Juppner and Schipani, 1996; Bro and Olgaard, 1997). Native 1–84 PTH and 1–34 PTH fragments stimulate adenylate cyclase through a GPCR with equivalent potency (Abou-Samra et al., 1992; Friedlander and Amiel, 1994; Blind et al., 1995; Bisello et al., 1996; Bro and Olgaard, 1997). PTH-related peptide (PTHrP) is a heterogeneous polypeptide with limited sequence homology to PTH in its amino terminal 13 residues. Both PTH and PTHrP can bind to and activate the same GPCR with equal affinity (Abou-Samra et al., 1989, 1994; Orloff et al., 1989; Amizuka et al., 2000a, 2000b). These receptors have been localized on embryonic chick and rabbit cartilage cells (Kioke et al., 1990). In chondrocytes, ligand binding of the type 1 PTH/PTHrP common receptor (PTH1R) activates several second-messenger systems with specific downstream phenotypic changes (Abou-Samra et al., 1994; Azarani et al., 1996; Suda, 1997; Serra et al., 1999; Kronenberg, 2006). Ligand binding to the common receptor PTH1R decreases the rate of growth plate cartilage differentiation (Vortkamp et al., 1996; Amizuka et al., 2000a). PTH1R gene knockout studies in mice have shown that this receptor binding was required for normal chondrocyte growth (Amizuka et al., 2002), differentiation, and bone development. Abnormal avian limb bud development occurred in prehypertrophic chondrocytes when the PTH/PTHrP receptor was repressed (Lanske et al., 1996; Amizuka et al., 2000b; Karaplis, 2001a). A stage-dependent activation of the PTH/PTHrP receptor in developing cartilage has been reported. Specifically, PTHrP represses terminal differentiation, holding chondrocytes in a prehypertrophic or proliferative stage. In addition, we demonstrated that PTH1R activation decreased apoptosis in cartilage, which can affect the total cell number (Harrington et al., 2004). The greatest number of apoptotic cells were in the cephalic hypertrophic region of control sterna (Hirsch et al., 1997b). The middle prehypertrophic and caudal proliferative regions of control sterna had significantly fewer apoptotic cells than the cephalic region. PTH-treated sternal cultures had a dose-dependent decrease in apoptosis in the hypertrophic cells but did not change in prehypertrophic or proliferative cells (Harrington et al., 2004). These results suggest that there may be regional differences in cell number in avian sternal cartilage. Chondrocytes from sterna cultured in the presence of 1011 and 107 M PTH contained larger cells in prehypertrophic and proliferative regions compared with control sterna. Our goal in the current project was to examine the effects of 100 nM PTH (1–34) on hyaline cartilage longitudinal growth. We examined two variables of growth measurement: changes in cell proliferation and changes in the extracellular matrix. Additionally, where possible, we examined regional differences. To accomplish this, a

serum-free avian organ culture model that recapitulates chondrogenesis to the hypertrophic stage (Hirsch and Svoboda, 1998) was used to investigate the following hypotheses. First, with a larger sample number, we tested the hypothesis that PTH1R activation significantly increased the longitudinal growth of the sterna on all days of the culture, not just the first half of the experiment as previously published. Additionally, we confirmed that PTH downregulated type X collagen deposition in the cephalic hypertrophic region, but also examined changes in the caudal region. Decreased type X collagen deposition served as a known histological marker of chondrogenic differentiation and confirmed that PTH ligand binding had successfully occurred. As such, it has become a critical first assay for all of the PTH-related studies. Second, we asked: Does 100 nM PTH (1–34) modulate cyclin d1 mRNA and Ki-67 production? Both proteins are known proliferation markers. Cyclins are a family of regulatory proteins that drive the ordered progression of mammalian cells through critical transition points in the cell division cycle. The D-type cyclins control the passage of cells through the G1 phase, allowing entry into the S phase. Cyclin D1 is a 295 amino acid, 34 kDa protein, known to be a target of PTHrP signaling (Datta et al., 2005). Furthermore, assessment of cell proliferation by detecting the Ki-67 antigen in cell populations has been used to determine a wider range of dividing cells. Ki-67 antigen is coexpressed during late G1, S, G2, and M phases, allowing for a wider range of proliferating cell populations than cyclin D1 (Lubke et al., 2005). Finally, we tested the hypothesis that 100 nm PTH (1–34) modulates the collagen content of the avian sterna, specifically types II, IX, and X. We asked if there were any observable regional changes. Types II and IX are usually expressed in the same tissue because type IX collagen is covalently linked to the larger type II collagen fibrils, and both are characteristic of cartilage, vitreous, and early avian cornea (Mayne, 1990; Hirsch et al., 1997a; Svoboda et al., 1998). Several studies, using cell lines or avian organ cultures, have reported a diminished response of collagen synthesis, both types II and X, with PTH1R activation (Loveys et al., 1993; Henderson et al., 1996; Zerega et al., 1999).

MATERIALS AND METHODS Organ Culture and Cartilage Preparation White Leghorn chicken eggs were obtained from Texas A&M Poultry Department. Whole day 14 sterna were dissected free of all tissue and perichondrial membranes under sterile conditions in Ham’s F-12 medium containing 1% antibiotic/antimycotic (Ab/Am; Gibco, Invitrogen, Grand Island, NY) and 1% nonessential amino acids (NEAA; Gibco, Invitrogen) (Hirsch et al., 1997a). Sterna were cultured for 8 days at 378C with 95% air/5% CO2 in a humid environment in sterile tissue culture chamber slides (Nunc) containing control medium (Ham’s F12 medium, 1% Ab/Am, 1% NEAA), 1011 M dexamethasone (Sigma Chemical, St. Louis, MO), 60 ng/ml insulin (Sigma), 1011 M triiodothyronine (T3; Collaborative Research), and 100 mg/ml ascorbic acid (Quarto et al., 1992). To determine the effects of PTH on cartilage growth, sterna were cultured with or without 100 nM PTH (Harrington et al., 2004). On the final culture day,

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Fig. 1. A: Twenty-four-hour growth rate was determined by measuring the sterna at the same time every day. PTH 100 nM group (black bars) grew at a significantly higher rate than controls (open bars; n ¼ 25). B: Means of cumulative sternal length. Total growth increased significantly in PTH 100 nM group (n ¼ 25). Triple asterisk, P < 0.001; double asterisk, P < 0.01; single asterisk, P < 0.05.

sterna were immediately processed for Ki-67, type II, IX, and X collagen immunohistochemistry, or cyclin D1, type II, IX, and X collagen mRNA quantitative RT-PCR. Sterna were also harvested on days 1 and 4 for Ki-67 immunohistochemistry.

Growth from Continuous Exposure to PTH Sterna were divided into two groups (n ¼ 8), controls and 100 nM PTH (bovine 1–34). The day the sterna were placed in culture was termed day 0. Media was changed daily and sternal length measurements were taken daily to determine overall growth on both PTHtreated and control samples for 8 days (Fig. 1A). In addition to overall growth, the rate of growth between groups was compared (Fig. 1B).

Immunohistochemistry for Ki-67 Deposition Sterna processed for Ki-67 immunohistochemistry were cut into three pieces (lateral plates and keel; Fig. 2). Chick sterna have two lateral plates with a perpendicular medial spine or keel (Hirsch et al., 1997a; Hirsch and Svoboda, 1998). After culturing, the spine and lateral plates were removed longitudinally along the medial keel, maintaining cephalic-caudal and medial-lateral orientations. The sterna were incubated for 1.5 to 3 hr in 0.1% testicular hyaluronidase (280 U/mg; Sigma) and washed in PBS. Tissues were then fixed in freshly prepared 4% paraformaldehyde for 15 min, rinsed in PBS, and blocked with 10% normal donkey serum (NDS; Life Technologies). The tissues were incubated in the Ki-67 primary antibody (Santa Cruz) overnight at room temperature on a rotary shaker. Ki-67 is an affinitypurified goat polyclonal antibody, dilution 1:50, directed against a peptide mapping near the C-terminus of mouse Ki-67. The primary antibody was visualized using Alexa-488 affinity-purified donkey antigoat IgG second-

ary antibody (Molecular Probes) diluted in 3% NDS and incubated at room temperature overnight. Following three rinses in PBS, the sterna keel and lateral plates were mounted in antifade mounting media (slowfade; Molecular Probes) on glass slides with nail polish spacers, coverslipped, and viewed on a Leica SP2 confocal laser scanning microscope. This whole mount procedure allowed mapping the different regions in a single specimen.

Quantification of Ki-67-Positive Cells Five plates from each group were randomly selected to count cells that stained positive for Ki-67. A total cell count on each plate was also conducted. Percentage of Ki-67-positive cells in a region was derived by dividing average number of positive cells by total cells counted. This entire process was completed independently by two examiners. Independent results had less than 5% variability across all groups. Both sets of results were averaged to obtain final cell numbers used for Ki-67 percentile calculation.

Immunohistochemistry for Type II, IX, and X Collagen Deposition Sterna processed for type II, IX, and X immunohistochemistry were cut into three pieces (lateral plates and keel), incubated for 1.5–3 hr in 0.1% testicular hyaluronidase (280 U/mg; Sigma), and washed in PBS to unmask the antigenic epitope (Schmid and Linsenmayer, 1985). Tissues were fixed in freshly prepared 4% paraformaldehyde (pH 7.4) for 15 min, rinsed in PBS, and blocked with 10% normal goat serum (NGS; Life Technologies). The tissues were incubated in type II, IX, and X collagen primary antibody (Schmid and Linsenmayer, 1985), dilution 1:100, overnight at room temperature on a rotary shaker. The primary antibody was visualized using

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Fig. 2. Regional PTH (100 nM) effects on type X collagen. Monoclonal antibodies specific for type X collagen were used to determine distribution of the secreted protein. In control sterna, type X collagen was deposited into the extracellular matrix surrounding hypertrophic chondrocytes (A) in the medial cephalic region (green). The nuclei were counterstained with ToPro (blue); type X collagen was decreased in sterna cultured in PTH 100 nM (B) compared to controls in the cephalic region. Both control (C) and PTH-treated sterna (D) had unde-

tectable type X collagen in the caudal region. The secondary antibody control was negative (E). F: A schematic diagram of the embryonic chicken sternum. This cartilage structure is characterized by a central spine region (keel) and two lateral plates. The areas of the three growth zones, hypertrophic, prehypertrophic, and proliferative, are illustrated and can be examined in a single lateral plate or keel. Scale bars ¼ 20 mM.

Alexa 488 affinity pure goat antimouse IgG secondary antibody (H þ L chains; Jackson ImmunoResearch) diluted in 3% NGS and incubated at room temperature overnight. Following three rinses in PBS, the sterna keel and lateral plates were mounted (slowfade; Molecular Probes) on glass slides with spacers, coverslipped, and viewed on Leica SP2 as described for Ki-67.

was used for each fluorescent tag scanned at the same confocal microscope settings with the opposite filter set and photomultiplier tube as crossover controls. Sterna incubated in the absence of a primary antibody did not demonstrate significant nonspecific binding of the secondary antibody (Hirsch and Svoboda, 1993, 1994; Hirsch et al., 1994).

Controls To determine background fluorescent staining, pieces of cartilage were incubated without primary antibody and incubated with a single-labeled secondary antibody. These samples were viewed with the Leica SP2 at the same settings as the experimental tissue. In addition to secondary antibody controls, it was necessary to demonstrate crossover between probes was not occurring. The Leica SP2 microscope has a subroutine that eliminated any possible overlap of signals. Single-labeled material

RNA Isolation/RT-PCR of Cyclin D1, Types II, IX, and X Collagen Whole day 14 sterna treated with or without PTH, were cultured for 8 days in a control medium and homogenized for RNA isolation using Tri-Azol (InvitrogenGibco, Carlsbad, CA). Using the National Center for Biotechnology Information (NCBI) Web site, four genes in chicken involved in early to late chondrogenesis were identified, cyclin d1, types II, IX, and X collagen (Table 1).

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TABLE 1. Representation of gallus-gallus genes and their designed primers used for RT-PCR

1 2 3 4

Protein

Primer-f

Primer-r

cyclin d1 Type X collagen Type II collagen Type IX collagen

aaacccaccttccatgat ggacaacagttttgcctg gggtggccatttagactt aatctggagagccaggac

attccttctcagatgccc gcccttagtgggaagtgt ccagacgtgcttcttgtc gggagaggagagaggtga

Primers for these genes and an internal control gene, 18S, were designed using Stratagene MX4000 PCR design software (Stratagene, La Jolla, CA). RNA was isolated for all treatment groups using Tri-Azol. A total of 100–1,000 ng of RNA was used for each RT reaction. For the MX4000 real-time PCR, a primer matrix was developed to determine the optimal combination of sense and antisense primer concentrations. Whole untreated sterna were used to establish the primer matrix, standard curves, and calibrations. The R2 values, slopes, and the efficiencies of each standard curve were recorded.

Confocal Laser Scanning Microscopy (CLSM) Fixed cartilage samples were analyzed with the Leica SP2 upright confocal scanning microscope. These images were optical sections through the vertical axis of the tissue. Tissue was viewed with a 403 water dipping lens (Leica). Confocal images were analyzed and stored. Color plates were arranged in Adobe Illustrator.

RESULTS Parathyroid Hormone (1–34) Increased Growth Rate and Cumulative Sternal Length Cultured sterna grew at an increased rate during all days of culture. For this experiment, the sample number was increased to 25 per group; this tripled previous experimental groups and yielded more informative growth statistics. On days 1–4, the difference in growth rate between groups was significant (triple asterisk, P < 0.001, Fig. 1A). On days 5–7, the growth rate was significantly different (double asterisk, P < 0.01). By day 8, significance was P < 0.05 (single asterisk), suggesting that although the growth rate was decreasing, PTH was still effective (Fig. 1A). Similarly, total growth statistical analysis demonstrated that the overall cumulative linear growth of the sterna treated with 100 nM PTH was significantly more than controls on all days of the culture, except day 0. On day 0, there was no difference in size between groups, indicating that all sterna started at the same developmental stage. On days 1–4, total sternal growth was significantly different (asterisk, P < 0.05). On day 5, significant differences increased to P < 0.01 (double asterisk). By days 6–8, the total sternal growth in the PTH 100 nM group was significantly different from controls (triple asterisk, P < 0.001, Fig. 1B).

Parathyroid Hormone (1–34) Regionally Decreases Type X Collagen Deposition The sternal organ culture model relies on two markers to establish that PTH1R was activated: an increase in total growth and a decrease in type X collagen deposition (Harrington et al., 2004). As in previous experiments, sterna cultured in control media had a continuous distri-

Fig. 3. Cyclin d1 mRNA RT-PCR quantifcation. Significant difference existed between the amount of dR (relative fluorescent amount) in controls and PTH-treated (P < 0.05) whole chick sterna.

bution of type X collagen (green) arranged in filamentous bundles surrounding lacunae containing cells (nuclei stained blue) in the medial cephalic region of the sterna (Fig. 2A). However, type X collagen staining intensity decreased dramatically with exposure to PTH 100 nM in the same region (Fig. 2B). Regionally, no detectable staining for type X collagen was observed in either the treatment or control groups in the caudal half (Fig. 2C and D). The secondary antibody controls were negative (Fig. 2E).

Parathyroid Hormone (1–34) Increased Cyclin d1 mRNA in Whole Sterna Organ Culture Cyclin d1 mRNA levels significantly increased (asterisk, P < 0.0122) in cartilage samples treated with PTH 100 nM (n ¼ 14) over controls (n ¼ 14; Fig. 3). The mean dR (fluorescent relative amount) levels of cyclin d1 mRNA in the PTH group were 0.468, compared to the dR of the control group: 0.369. R2 values of 0.965 and 0.981 were obtained on the MX-400 for the standard curves of 18s and cyclin D1 (not shown). There were equal amounts of 18s internal RNA in all samples.

Parathyroid Hormone (1–34) Regionally Modulates Ki-67 in Avian Sterna Cyclin d1 increases in PTH-treated sterna suggested only global cell proliferation because the mRNA extraction protocol required whole sternal homogenization.

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Fig. 4. Control sterna with (A) Ki-67 proliferation marker (green), (B) actin labeled with phalloidin (red), (C) and nuclei labeled ToPro (blue). D: Merged image demonstrating that proliferating cells were on the periphery and Ki-67 was detected in the cytoplasm and nuclei.

to M phase and therefore would label more proliferating cells than cyclin d1. We used this histological marker to test for the regional and temporal effects of PTH. Baseline immunohistochemistry with Ki-67 (green), actin phalloidin (red), and nuclei using ToPro (blue) was completed to determine the appearance of samples when all three markers were positive (Fig. 4A–D). In the cephalic region, after 1 day of PTH treatment, 36.65% of the PTH-treated chondrocytes were positive for Ki-67 (Figs. 5A, black bars, and 6D) compared to 16.92% of controls (Figs. 5A, open bars, and 6A). On day 4, 33.46% of the PTH treatment group had Ki-67 (Figs. 5A, black bars, and 6E) compared to 16.08% in controls (Figs. 5A, open bars, and 6B). On day 8, the PTH groups had 36.82% Ki-67 staining (Figs. 5A, black bars, and 6F) compared to 3.04% in controls (Figs. 5A, open bars, and 6C). A one-way ANOVA test determined that there was a significant difference between the PTH-treated group and controls in the amount of Ki-67 detection (P < 0.001). A two-way ANOVA analysis confirmed the interaction between the effects of PTH and changes in Ki-67-labeled cells temporally over the 8-day culture (P < 0.001). In the caudal region, on day 1, 18.18% of the PTH-treated chondrocytes were labeled with Ki-67 (Figs. 5B, black bars, and 6J) compared to 43.03% of controls (Figs. 5B, open bars, and 6G). On day 4, 1.8% of the PTH treatment group had Ki-67 (Figs. 5B, black bars, and 6K) compared to 3.2% in controls (Figs. 5B, open bars, and 6H). On day 8, the PTH groups had 6.8% Ki-67 staining (Figs. 5B, black bars, and 6L) compared to 17.87% in controls (Figs. 5B, open bars, and 6I). A one-way ANOVA analysis determined that there was a significant difference between the PTH treatment group and controls on each separate day (P < 0.001). However, two-way ANOVA analysis shows no consistent relationship, regionally or temporally, between treatment of PTH 100 nM and Ki-67 detection.

Fig. 5. PTH (100 nM) increased Ki-67 in hypertrophic chrondrocytes but not proliferative chrondrocytes. A: Cephalic cells with Ki-67positive staining (graph) and the total number of cells counted (Table 1). Positive cell counts were repeated on days 1, 4, and 8. A significant difference (P < 0.0001) between controls (open bars) and PTH-treated sterna (black bars) was found on all 3 days. Two-way ANOVA analyses

also had a significant interaction between culture day, PTH, and Ki-67 in the cephalic region. B: Caudal region. A significant difference (P < 0.0001) was shown between controls (open bars) and PTH treatment groups (black bars) on all 3 days. Two-way ANOVA analysis in the caudal area, however, did not show interaction between culture day, treatment of PTH 100 nM, and Ki-67.

Investigating further, we wanted to determine if there was a region of the sterna more proliferation-sensitive to PTH, and whether the PTH exposure created a temporal relationship with cell number during the organ culture. The Ki-67 protein should be expressed in cells from G1

Fig. 6. Regional distribution of Ki-67 (green), actin (red), and nuclei (blue) in cephalic (A–F) and caudal (G–L) regions. PTH-treated cartilage after day 1 (A, D, G, and J), day 4 (B, E, H, and K), and day 8 (C, F, I, and L). Control sterna (A–C, G–I) were compared to PTH treated sterna (D–F, J–L). Ki-67 was located in the cytoplasm (A, D–I) and

around nuclei. Control sterna had more Ki-67-positive cells in the caudal (G–I) than cephalic region (A–C). In contrast, the PTH-treated sterna had more Ki-67-positive cells in the cephalic (D–F) region than either the control (A–C) or caudal region (J–L). Scale bars ¼ 20 mM.

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Fig. 7. Collagen mRNA RT-PCR quantifcation. A: PTH (black bars) decreased type II collagen mRNA determined by dR (relative fluorescent amount) compared to controls (P < 0.05). B: In contrast, type IX collagen mRNA increased in the presence of PTH (black bars) compared to controls (open bars; P < 0.05). C: Type X collagen mRNA (black bars) also decreased in PTH-treated sterna (P < 05), confirming previous studies (Harrington et al., 2004). Whole sterna were used in these experiments.

Parathyroid Hormone (1–34) Modulated Type II, IX, and X Collagen Cartilage can also change through extracellular matrix deposition. Therefore, we determined if the collagen profile changed in the presence of PTH. Type IX mRNA levels increased (P < 0.05) in cartilage samples treated with PTH 100 nM (n ¼ 14) over controls (n ¼ 14; Fig. 7B). The mean dR (fluorescent relative amount) levels of type IX collagen mRNA (Fig. 7B) in the PTH group were 0.718, compared to the dR of the control group: 0.192. Types II and X collagen decreased (P < 0.05) in cartilage samples treated with 100 nM PTH (1–34) compared to controls (Fig. 7A and C). The mean dR (fluorescent relative amount) levels of type II collagen mRNA (Fig. 7A) in the PTH group were 1.343, compared to the dR of the control group: 3.025. The mean dR (fluorescent relative

amount) levels of type X collagen mRNA (Fig. 7C) in the PTH group were 1.342, compared to the dR of the control group: 2.384. The experiment was performed in triplicate. R2 values of 0.965, 0.968, and 0.929 were obtained on the MX-4000 for the standard curves of 18s and type II and IX collagen (not shown). There were equal amounts of 18s internal RNA in all samples. These results were consistent with immunohistochemical observations of type II collagen. PTH-treated sterna had less type II collagen in the cephalic region (Fig. 8A) than controls (Fig. 8B). In the caudal region, there was less discrepancy between culture conditions, with a slight decrease in type II collagen in the PTH treatment group (Fig. 8C) compared to controls. Increased cell size and number were observed in the PTH-treated caudal area compared to controls.

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Fig. 8. Regional IHC of types II and IX collagen treated with PTH (100 nM). Type II collagen (green), nuclei (blue): (A) PTH-treated, cephalic; (B) control, cephalic; (C) PTH-treated, caudal; (D) control, caudal. Type IX collagen: (E) PTHtreated, cephalic; (F) control, cephalic; (G) PTH-treated, caudal; (H) control, caudal. Note (yellow arrowheads) in both treatment and control caudal samples, there was extracellular staining of type IX collagen directly at the cell surface, suggesting initial collagen formation. Scale bars ¼ 40 mM.

Immunohistochemistry of type IX collagen (Fig. 8E–H) revealed differences between the cephalic (Fig. 8E–F) and caudal region (Fig. F and G). In the cephalic region, in both treatment and control samples, the type IX collagen was present in the extracellular matrix, but with less staining detected in the PTH treatment group. In the caudal area, type IX collagen appeared trapped in the lacunae (Fig. 8G and H, arrowhead), directly outside the cell, perhaps at the cell surface, suggesting initial collagen formation in both groups.

DISCUSSION Determining the factors that contribute to craniofacial abnormalities is our long-term goal. In the craniofacial complex, primary cartilage contributes to the growth of the midface in the nasal septum and cranial base. Unlike many primary cartilage studies that use the long bone as a model, we have chosen the well-studied avian sterna, which is analogous to the non–load-bearing craniofacial structures.

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The PTH/PTH receptor is integral for normal growth of primary cartilage. In humans, a double mutation in the receptor gene is lethal. A single copy mutation may result in Jansen’s metaphyseal chondrodysplasia (JMC). JMC is a rare autosomal dominant form of short limb dwarfism characterized by asymptomatic hypercalcemia and skeletal deformities, with low PTH and PTHrP levels (Minagawa et al., 1997; Schipani and Provot, 2003; Bastepe et al., 2004). This rare disorder may be caused by activating mutations in the PTH/PTHrP receptor, leading to ligand-independent cAMP accumulation. Analysis of genetically altered mice that lack either PTHrP or the PTH/PTHrP receptor, and transgenic mice that have mutant receptors targeted to the growth plate, has provided a molecular explanation for the severe skeletal abnormalities seen in JMC (Amizuka et al., 2002). Studies of JMC have elucidated the fundamental role of PTH/PTHrP receptors in mediating the paracrine/autocrine actions of PTHrP in growth plate development, bone elongation, and PTH endocrine actions (Kronenberg et al., 1998; Kronenberg, 2006). Several studies in other tissues have reported a mitogenic or proliferative effect from increased ligand binding to this receptor (Karaplis, 2001b; Higashikawa et al., 2006). PTHrP may be a potential paracrine factor acting via the PKA pathway in the proliferation and formation of endothelial cells in malignant pituitary tumors (Akino et al., 2000). PTHrP (38–64) stimulated lung type II cell growth and may have a role in lung repair in silicainjured rats (Hastings et al., 1994). PTHrP was also a modulator of pancreatic islet growth and/or function by a PKC-mediated mechanism (Villanueva-Penacarrillo et al., 1999). In the secondary cartilage of the mandibular condyle, there was evidence that condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP played a pivotal role in regulating the proliferative capacity and differentiation of these cells (Suda et al., 1999; Ng et al., 2006). Variables that may contribute to the tissue growth include cell number, cell size, and components of the extracellular matrix, collagen, and proteoglycans, such as aggrecan. We have previously reported the effects of PTH on cell size in growing cartilage, concluding that chondrocytes from sterna cultured in the presence of PTH contained larger cells in prehypertrophic and proliferative regions, leading to the question of whether the independent mechanisms of growth should be investigated (Harrington et al., 2004). Additionally, we have shown that PTH modulated apoptosis in the avian sterna. The cephalic-hypertrophic region of control sterna had significantly higher numbers of apoptotic nuclei than the middle prehypertrophic and caudal proliferative regions. However, sterna cultured in the presence of PTH had a dose-dependent decrease in apoptosis in the hypertrophic cells but did not change in prehypertrophic or proliferative cells (Harrington et al., 2004), once again highlighting that there may be a regional component to be discussed in the overall growth model. In this study, we attempted to confirm through more reliable growth data, proliferation (cyclin d1, ki-67, ECM content assays, collagen mRNA quantification, and IHC), and regional observations that 100 nm PTH (1–34) modulates primary cartilage growth in order to answer specifically when, how, and where the growth was occurring.

First, we were able to determine, by tripling previous sample sizes, that significant growth of the sternal cartilage did not occur uniquely during the initial days of exposure to PTH, as previously published, but continued to grow significantly along the longitudinal axis throughout the entire 8-day culture, a serum-free organ culture model that recapitulated in ovo chondrogenesis to the hypertrophic stage in the cephalic region. This model was used because it maintains the extracellular chrondrocyte milieu. In addition, PTH-treated cartilage maintained a significantly higher growth rate through each day of the culture. Although both the growth rate and overall longitudinal measurements were attenuated toward the end of the culture, we propose that PTH may play a contributing role throughout all stages of cartilage growth and differentiation instead of a focal initial influence. Second, previous studies have implicated cyclin protein involvement in chondrogenesis, specifically PTHmediated chondrogenesis (MacLean et al., 2004; Datta et al., 2005; Qin et al., 2005). Chondrocyte proliferation and differentiation requires cellular attachment to the collagen type II-rich matrix. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). Inactivation of both copies of the ILK allele has led to an additional chondrodysplasia characterized by a disorganized growth plate and to dwarfism, such as JMC. Expressions of chondrocyte differentiation markers such as types II and type X collagen, Indian hedgehog, and the PTH-PTHrP receptor were normal in ILK-deficient growth plates. However, chondrocyte proliferation assessed by BrdU or proliferating cell nuclear antigen PCNA labeling was markedly reduced in the mutant growth plates. Cell-based assays showed that the integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. Interestingly, ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explained the defect in chondrocyte proliferation (Terpstra et al., 2003). Additionally, previous studies reported that JMC mutations of the PTH/PTHrP receptor induced activation of the cyclin D1 and cyclin A promoters in primary mouse chondrocytes and rat chondrosarcoma cells (Beier and LuValle, 2002). JMC mutations were characterized by a constitutively active PTH/PTHrP receptor, similar to our daily exposure of PTH treatment in sterna. Their data suggested that stimulation of cell cycle gene expression and cell cycle progression by mutant PTH/PTHrP receptors contributes to the pathogenesis of JMC. In this experiment, it was determined that PTH regulated proliferation by increasing cyclin d1 mRNA. The histological proliferation marker Ki-67 was used to determine regional and temporal differences between groups. Samples from the cephalic and caudal regions at three time points were analyzed. In the cephalic region, on all days (1, 4, 8), the sterna treated with PTH had significantly higher percentages of Ki-67-positive cells. A two-way ANOVA analysis confirmed that there was a significant interaction between day of culture, treatment, and Ki-67-positive cells. Hence, If there was no interaction overall, there is a less than 0.01% chance of randomly observing so much interaction in an experiment of this size. The interaction was considered significant in the analyses and suggests a temporal relation-

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ship between PTH exposure and proliferation in the cephalic region. In the caudal region, however, there was variation in the percentage of Ki-67-positive cells at the three time points, with day 4 sterna having the lowest percentage of positive Ki-67-labeled cells. Additionally, the control group demonstrated more Ki-67-positive cells than the PTH-treated groups. Although a difference in Ki-67 between treatment and control groups was found, statistical analysis indicated that PTH did not have an interaction with Ki-67-positive cells over the course of the organ culture. Studies have supported the theory that the number of PTH/PTHrP receptors on cell surfaces can be modulated by external physiological conditions (Lee et al., 1993; Urena et al., 1996). We hypothesize that the number of receptors is reduced in the caudal region; therefore, PTH had little to no consistent effect on cell proliferation. Finally, to test whether there was a change in ECM content from exposure to PTH, we designed primers for types II, IX, and X collagen. We observed significant decreases in type II collagen mRNA but not its associated collagen, type IX. As expected and previously reported, we confirmed that type X collagen mRNA decreased from PTH treatment. Noticeable regional changes in collagen deposition were detected from immunochemistry in all types (II, IX, and X) of collagen. Two noteworthy observations were the diminished detection of type II collagen in the cephalic PTH treatment group compared to controls, supporting the mRNA results and type IX collagen detection on the cell surface of chondrocytes, suggesting initial collagen formation in the caudal areas of both conditions. As reported in other studies, using different models, collagen synthesis decreased in a dose-dependent manner in the presence of PTH (Loveys et al., 1993; Henderson et al., 1996; Zerega et al., 1999). In chick tibia, it was reported that PTH had a stimulatory effect on proliferation of chondroprogenitor cells and inhibited collagen and matrix synthesis in the epiphyseal cartilage via cAMP-dependent pathways (Pines et al., 1990). An important extracellular matrix protein that is missing from this analysis, but should be included in future studies, is aggrecan. Aggrecan is the shortened name of the large aggregating chondroitin sulfate proteoglycan. Aggrecan, one of the most widely studied proteoglycans, is abundant in primary cartilage, representing 10% of the dry weight of cartilage (cartilage is up to 75% water). The effects of PTH on aggrecan have previously been reported in different models. For example, chondrocytes isolated from the rib cage of developing rat embryos were evaluated for the biosynthesis of aggrecan. Cells treated with PTH (1–34) showed a significant dose-dependent increase of aggrecan mRNA synthesis (Harvey et al., 1999). In another study, cells derived from the clonal chondrocytic cell line, CFK2, increased expression of mRNA for type II collagen, aggrecan, and link protein. Interestingly, this same study also concluded that the PTHrP ligand may act as a bifunctional modulator of cartilage growth and subsequent receptor binding may signal a mechanism distinct from the classical signal transduction pathways linked to the PTH/PTHrP receptor (Henderson et al., 1996). This observation, coupled with an emerging large body of cross-talk literature, may help explain the mixed results found here with respect to collagen levels.

The question as to whether the mRNA and protein deposition levels of aggrecan are changed in response to PTH treatment is important. Additionally, as aggrecan is the major water attractor in cartilage, conclusions about the levels of aggrecan will suggest changes in the tissue’s diffusion capability. Such data, combined with the collagen results, would provide a more comprehensive answer to how changes in matrix synthesis contributes to the changes in cartilage growth in response to PTH treatment. In summary, 100 nM PTH (1–34) modulated primary cartilage growth in the avian sternal model. Combined results demonstrated that different regions of the sterna appeared to contribute different growth dynamics. In the caudal region, cell size in the PTH-treated cartilage was significantly larger than controls (Harrington et al., 2004). However, growth appeared to originate in the cephalic region. Increased cell proliferation and decreased apoptosis in the cephalic region supports the theory that this sterna region contributes to the overall growth in PTH-treated sterna. Diminished collagen synthesis in PTH-treated sterna was an important variable but did not overcome the increased proliferation or decreased apoptosis in the cephalic region. As mentioned, further studies should be conducted into the role of several other extracellular matrix components, including proteoglycans, particularly aggrecan. We could not conclude that collagen deposition alone was related to the changes in growth, whereas cell proliferation has emerged as a contributing factor in differences in longitudinal growth observed in cartilage exposed to parathyroid hormone. Consequently, if cell proliferation could be modulated in response to PTH, this may have remarkable implications and applications for future studies into fracture repair and/or replacement of damaged or genetically dysfunctional cartilage.

ACKNOWLEDGMENTS Supported by the National Institutes of Health/F30 DE05743 (E.K.H.) Training Grant and the National Institutes of Health/National Institute of Dental and Craniofocial Research T35 Grant. The authors thank Sala Senkayi for technical assistance, and Drs. Mihir Patel, Matthew Roberts, and Paul Denson for support during their summer research projects.

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