help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0695
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/10/5030    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woods, A.
Right arrow Articles by Beier, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woods, A.
Right arrow Articles by Beier, F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 148, No. 10 5030-5041
Copyright © 2007 by The Endocrine Society

C-Type Natriuretic Peptide Regulates Cellular Condensation and Glycosaminoglycan Synthesis during Chondrogenesis

Anita Woods, Sameena Khan and Frank Beier

Canadian Institutes for Health Research Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1

Address all correspondence and requests for reprints to: Frank Beier, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: fbeier{at}uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C-type natriuretic peptide (CNP) has recently been identified as a key anabolic regulator of endochondral bone growth, but the cellular and molecular mechanisms involved are incompletely understood. Although CNP has been shown to stimulate proliferation and hypertrophic differentiation of growth plate chondrocytes, it is unknown whether CNP affects the earliest stages of endochondral bone development, condensation of mesenchymal precursor cells, and chondrogenesis. Here we demonstrate that CNP increases the number of chondrogenic condensations of mouse embryonic limb bud cells in micromass culture. This is accompanied by increased expression of the cell adhesion molecule N-cadherin. In addition, CNP stimulates glycosaminoglycan synthesis as indicated by increased Alcian blue staining. However, expression of the chondrogenic transcription factors Sox9, -5, and -6 or of the main extracellular matrix genes encoding collagen II and aggrecan is not affected by CNP. Instead, we show that CNP increases expression of enzymes involved in chondroitin sulfate synthesis, a required step in the production of cartilage glycosaminoglycans. In summary, we demonstrate a novel role of CNP in promoting chondrogenesis by stimulating expression of molecules involved in cell adhesion molecules and glycosaminoglycan synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C-TYPE NATRIURETIC PEPTIDE (CNP) is a member of the natriuretic peptide family, together with the related ANP and BNP proteins (1). CNP is found in many tissues, including cartilage (2), and signals through natriuretic peptide receptor 2 (Npr2). A second receptor for CNP, Npr3, is thought to act as a decoy/scavenger receptor that limits CNP effects by removing it. Npr3–/– mice display skeletal overgrowth, similar to that seen in BNP-overexpressing mice (3). In contrast, disruption of the genes encoding CNP (Nppc) or Npr2 results in severe dwarfism and impaired endochondral ossification (4, 5). Targeted expression of CNP in growth plate chondrocytes can rescue the phenotype of Nppc–/– mice (6). Importantly, loss-of-function mutations in the human NPR2 gene result in idiopathic short stature when heterozygous and acromesomelic dysplasia, type Maroteaux, when homozygous (7, 8). In contrast, ectopic CNP has been show to rescue growth retardation in a mouse model of achondroplasia, suggesting significant therapeutic potential for CNP in the treatment of dwarfism (9). Previous studies demonstrate that CNP expression occurs in developing cartilage and acts locally as a positive regulator of endochondral ossification through the accumulation of cGMP by NPR2 (10). cGMP activates cyclic nucleotide phosphodiesterases, cGMP-regulated ion channels, and cGMP-dependent protein kinases. CNP and cGMP have been determined to stimulate longitudinal growth of long bones (11, 12). However, although CNP has been shown to promote chondrocyte proliferation and late (hypertrophic) differentiation, a role for CNP in early chondrogenesis has been suggested, but not described (13).

Endochondral ossification is the developmental process that creates the axial and appendicular skeleton, beginning with the initial step termed chondrogenesis (14). Mesenchymal cells are stimulated to condense, resulting in increased cell-cell interactions and increased cell density without an increase in proliferation (15). The size, location, and shape of condensations directly determines the formation of the intermediary cartilage template from which endochondral bone is laid down (16, 17).

Condensing mesenchymal cells express the cell adhesion molecules N-cadherin and N-CAM (18, 19, 20, 21) and the disaccharide galactosidase(ß1,3) N-acetyl galactosamine, which can be visualized by staining with the lectin peanut agglutinin (PNA) (22). The mesenchymal cells within the condensations are stimulated to differentiate into chondrocytes, the cell type of cartilage (15). Chondrocytes are responsible for generating and maintaining the cartilaginous extracellular matrix (ECM), providing tensile and compressive strength in articular cartilage, and providing the scaffold on which the future bone is laid down (23, 24). Therefore, identification of signaling pathways that regulate chondrocyte differentiation and chondrocyte matrix production are vital to both the understanding of skeletal development and for advancement of therapeutics for diseases such as osteoarthritis.

The transcription factor Sox9 is expressed in condensed mesenchyme and is required for chondrogenic differentiation (25, 26). Also necessary for chondrocyte differentiation are the transcription factors Sox5 and -6, which together with Sox9 regulate the expression of the matrix molecules collagen II and aggrecan (27, 28, 29). Collagens interact with other matrix components such as the proteoglycan aggrecan (30, 31). Aggrecan is the major core proteoglycan found in the chondrocyte ECM, aggregates with other ECM components (32, 33, 34), and is heavily modified posttranslationally by sulfated glycosaminoglycans (GAGs), giving the aggregate a highly negative charge, detectable by the tetracationic dye Alcian blue (35, 36). The attraction of numerous water molecules results in provision of compressive strength to the matrix, and these aggregates also function to regulate and immobilize growth factors found within the matrix (36, 37). Link protein functions to stabilize the formation of the aggregates (38).

GAGs are long unbranched polysaccharide chains made up of repeating disaccharide units that can be covalently linked to specific amino acids of aggrecan (36), beginning with addition of xylose that is catalyzed by xylosyltransferases (39). Xylosyltransferases I and II are expressed in chondrocytes and are necessary for the addition of GAGs to the core protein (40, 41, 42). GAGs are then sulfated by sulfotransferases to attach sulfate groups to specific sugar moieties (43). A chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities (44). The sulfotransferases are developmentally regulated and involved in cell adhesion and migration, for example in neurons (45). Deletion of chondroitin 6-sulfotransferase 3 (Chst3) does not result in a skeletal phenotype in mice; however, a mutation in this gene does result in a chondrodysplasia in humans (46). In contrast, inactivation of the chondroitin 4-sulfotransferase 11 (Chst11) gene results in severe disturbance of endochondral ossification in mice (47). However, the signals and mechanisms that regulate the expression of genes involved in GAG synthesis during chondrogenesis are largely unknown.

Here we demonstrate that CNP is an important regulator of chondrogenesis through the regulation of mesenchymal condensations and matrix biosynthesis. In particular, CNP treatment results in an increased expression of transcripts for N-cadherin, link protein, xylosyltransferase I, Chst11, and Chst3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Timed pregnant CD1 mice (at 11.5 d postcoitum) were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). All cell culture reagents were from Invitrogen (Burlington, Ontario, Canada) unless stated otherwise. CNP (catalog no. N8768) and 8-(4-cpt) cGMP (catalog no. C5438) were purchased from Sigma Chemical Co. (St. Louis, MO). General chemicals were from Sigma or Calbiochem (La Jolla, CA) unless otherwise stated. The SOX9 reporter plasmid was a generous gift from Dr. M. Underhill (University of British Columbia). The collagen II antibody (catalog no. ab-21291) was purchased from Abcam (Cambridge, MA), and the aggrecan antibody (catalog no. AF-1220) was purchased from R&D Systems (Minneapolis, MN).

Micromass culture
Mesenchymal cells were isolated from the limbs of embryonic d-11.5 mice as described (48, 49). Cells were resuspended in media containing 60% F12, 40% DMEM, 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD), 0.25% penicillin/streptomycin, and 0.25% L-glutamine and plated in 10-µl droplets at a density of 2.5 x 107 cells/ml in six-well plates (Grenier Bio-One, Frickenhausen, Germany). After 1 h, cells were fed with media containing BSA/HCl for controls, 0.01–1 µM CNP (Sigma), or 100 µM 8-(4-cpt) cGMP and supplemented with 1 mM ß-glycerolphosphate and 50 µg/ml ascorbic acid. Media and treatments were replaced every 24 h for a period of 6 d.

Real-time RT-PCR
RNA was isolated on d 1, 3, and 6 of micromass culture with an RNeasy kit (QIAGEN, Valencia, CA) as described per the manufacturer’s protocol. Twenty-five nanograms of total RNA were plated per well in quadruplicate. Relative gene expression was determined by measuring Npr2, Npr3, N-cadherin, N-CAM, Sox9, Sox5, Sox6, collagen II, aggrecan, link, Chst3, Chst11 (Assays on Demand; Applied Biosystems, Foster City, CA) relative to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) using one-step RT qPCR Master Mix (Eurogentec, Liège, Belgium) and 40 cycles on the ABI prism 7900 HT sequence detector (PerkinElmer Life Sciences, Norwalk, CT).

Alcian blue staining and quantification
Micromass cultures were isolated on d 1, 3, and 6 of culture and fixed in 100% ethanol for 20 min at –20 C. Cultures were incubated with 0.1% HCl-Alcian Blue solution for 2 h at room temperature and then rinsed in double-distilled water (49). Pictures were taken, and then stain was solubilized with 6 M guanidine hydrochloride for 8 h at room temperature. Absorbance was measured with a spectrophotometer at 620 nm and normalized to protein or RNA content. Protein was isolated from parallel wells in RIPA lysis buffer supplemented with protease inhibitors and quantified by bicinchoninic acid (Sigma) as described by the manufacturer’s protocol (49).

PNA staining
Micromass cultures were fixed in 4% paraformaldehyde at room temperature for 30 min. Cultures were then washed in PBS and incubated for 2 h with 50 µg/ml horseradish peroxidase-conjugated PNA diluted in PBS. PNA was detected colorimetrically with diaminobenzidine (Dako, Carpinteria, CA). Excess stain was rinsed of with double-distilled water, and bright-field images were taken. Protein was isolated from parallel wells in RIPA lysis buffer supplemented with protease inhibitors and quantified by bicinchoninic acid (Sigma) as described above. The number of PNA-stained nodules was counted by an independent observer unaware of experimental conditions and normalized to protein content.

Transient transfection
Mesenchymal cells were resuspended in micromass media at a density of 2.5 x 107 cells/ml and transfected with Fugene6/DNA in a ratio of 1:1 with 0.5 µg plasmid DNA containing a Sox9 reporter linked to a firefly luciferase gene (50) and 0.5 µg of a plasmid containing the SV40 promoter linked to renilla luciferase gene (Promega, Madison, WI). After incubation for 15 min, cells were plated as micromass cultures and treated as above. Cells were harvested on d 3 of culture, and lysates were measured for luciferase activity with the dual-luciferase kit (Promega) as described by the manufacturer’s protocol. Relative luciferase activity was measured as described previously (49).

Immunohistochemistry
Micromass cultures were isolated on d 1, 3, and 6 of culture and fixed in 4% paraformaldehyde at room temperature for 30 min. Cultures were incubated with 0.1% Triton X-100/PBS for 10 min and rinsed in PBS. Cultures were blocked in goat or rabbit serum diluted 1:20 in PBS for 30 min at room temperature. After 1 h incubation with anticollagen II or antiaggrecan antibodies (1:200 in blocking solution), cultures were washed in PBS and then incubated for an hour with appropriate secondary antibodies conjugated to horseradish peroxidase. Micromass cultures were rinsed with PBS, and proteins were detected by diaminobenzidine. Bright-field images were taken at a x4 magnification.

Statistical analysis
Data collected from Alcian blue quantification and nodule counts are the average of three independent experiments run in duplicate. Means were quantified relative to total protein and normalized to the vehicle control. Data collected from real-time PCR are an average of three independent trials of samples run in quadruplicate. Means were quantified relative to Gapdh, and then data were normalized to d 1 of control treated RNA per trial. Statistical significance was determined by a one- or two-way ANOVA with Bonferroni post test using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CNP receptors are expressed and up-regulated during chondrogenesis
We first investigated the expression of the CNP receptor during early chondrogenesis. RNA was isolated from micromass cultures over a period of 6 d in culture. Real-time PCR analyses revealed that Npr2 is expressed on d 1 of culture, but expression increased 70-fold by d 6 of chondrogenic culture (Fig. 1AGo). The CNP decoy receptor Npr3 also displayed marked up-regulation of transcript levels during chondrogenesis but not as dramatic as Npr2 (Fig. 1BGo).


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 1. The CNP receptors Npr2 and Npr3 are expressed during chondrogenesis. RNA was isolated from micromass cultures over a period of 6 d. A, Real-time PCR demonstrates that Npr2 expression increases with time in vehicle control cultures; B, the gene encoding the decoy receptor, Npr3, displays a similar expression patterns, but its induction is not as dramatic as for Npr2. Data shown are the average means of three independent trials run in quadruplicate. *, P < 0.05.

 
CNP signaling stimulates GAG accumulation
We next examined whether CNP regulates chondrogenesis. Micromass cultures were incubated with BSA/HCl (vehicle) or 10 nM, 100 nM, or 1 µM CNP and then stained with Alcian blue to assess GAG accumulation, a well-documented marker of chondrogenic differentiation. Increasing concentrations of CNP resulted in a visual increase in the number of Alcian blue-stained nodules (Fig. 2AGo). Extraction and photometric quantification of Alcian blue staining confirmed that CNP increases GAG synthesis in a concentration-dependent manner (data not shown). Alcian blue staining of micromass cultures in a time course of d 1, 3, and 6 demonstrated an increase in Alcian blue stain over time (Fig. 2BGo). Cultures treated with 1 µM CNP showed an increased Alcian blue staining compared with control cultures on d 6 (Fig. 2BGo). Alcian blue stain was solubilized and quantified and then normalized to total protein, demonstrating increasing Alcian blue stain over time in culture and also a significant increase of stain resulting from CNP treatment on d 6 of culture (Fig. 2CGo). Quantified Alcian blue stain was also normalized to total RNA and also demonstrated similar significant increases with CNP treatment compared with control cultures (data not shown).


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		FIG. 2. CNP signaling stimulates mesenchymal condensations and GAG production. A and B, Mouse embryonic mesenchymal limb bud cells were plated in micromass culture and incubated in the presence of vehicle or CNP at the indicated concentrations for 6 d (A) or with 1 µM CNP for the indicated time (B); C, cells were stained with Alcian blue. Alcian blue stain was extracted from cultures in B, quantified by spectrophotometry, and normalized to total protein. These data demonstrate that GAG accumulation significantly increases with time and that CNP stimulates GAG synthesis in comparison with control cultures. Data shown are representative of at least three independent trials (A and B) or are the average of three independent trials ± SEM (C). *, P < 0.05.

 
CNP promotes cellular condensation and N-cadherin expression
PNA staining is used to visualize condensing mesenchyme. On the first day of micromass culture, cells stained with PNA, and the stain became more intense and increasingly more nodular by d 6 of culture (Fig. 3AGo). Manual counts demonstrated an approximately 30% increase in the number of stained condensations on d 6 in CNP-treated cultures normalized to total protein (Fig. 3BGo), confirming that the CNP signaling pathway stimulates the formation of condensations. Absolute number of condensations and condensation number normalized to total RNA showed similar significant increases in response to CNP treatment (data not shown). N-cadherin and N-CAM are important cell adhesion molecules regulating mesenchymal condensations in chondrogenesis. Treatment of cultures with CNP resulted in a significant increase of N-cadherin transcript levels by d 6 of culture as well as an increase (although not significant) in N-CAM mRNA (Fig. 3Go, C and D).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 3. CNP signaling stimulates mesenchymal condensations. A, Micromass cultures grown in the presence of 1 µM CNP or BSA/HCl control were incubated with PNA, and condensations were visualized by PNA stain; B, the number of cellular condensations were counted by an independent observer and normalized to protein content, and results show that CNP treatment increases the number of mesenchymal condensations. C, Real-time PCR demonstrates a significant increase in the mRNA levels of N-cadherin by d 6 of culture; D, although CNP treatment increased mRNA levels of N-CAM, these results were not significant. Data shown are representative (A) or the average of at three independent trials ± SEM (B–D). *, P < 0.05.

 
cGMP treatment increases chondrogenesis similarly to CNP treatment
We next asked whether we could mimic the effects of CNP signaling by treating cells with an analog of cGMP. Micromass cultures treated with CNP or cGMP demonstrated a similar increase in Alcian blue (Fig. 4AGo) and PNA (Fig. 4BGo) stains. The number of condensations was counted, showing that both CNP and cGMP give rise to significantly more nodules than control cultures relative to total protein (Fig. 4CGo). The absolute number of nodules and the number of nodules relative to total RNA also demonstrated similar significant increases in response to CNP and cGMP treatment in comparison with controls (data not shown). These data suggest that the effect of CNP signaling is likely occurring through increasing intracellular levels of cGMP.


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 4. cGMP treatment mimics CNP effects on GAG accumulation and cellular condensations. A, Cultures were treated with vehicle, 100 µM 8-(4-cpt) cGMP, or 1 µM CNP for a period of 6 d and then stained with Alcian blue. cGMP and CNP treatment demonstrate a similar increase of GAG accumulation in comparison with control cultures, after normalization to protein content; B, micromass cultures were treated with vehicle, 1 µM CNP, or 100 µM 8-(4-cpt) cGMP for a period of 6 d and then incubated with PNA, and both CNP and cGMP treatment result in similar increases of stained nodules; C, the number of cellular condensations from B were counted by an independent observer and normalized to protein content, showing that cGMP and CNP treatment both result in significantly increased numbers of condensations compared with controls, relative to total protein. Because both CNP and cGMP treatments result in similar effects, this suggests a similar pathway of action. Data shown in A and B are representative of three independent experiments and in C are the average mean ± SEM of three independent experiments run in duplicate. *, P < 0.05.

 
CNP signaling promotes chondrogenesis in a Sox9-independent manner
The transcription factor Sox9 is a central and essential regulator of chondrogenesis (51). We therefore asked whether CNP signaling stimulates chondrogenesis through stimulation of Sox9 expression and/or activity. However, real-time PCR showed that CNP treatment did not induce significant changes in the levels of Sox9 mRNA in micromass cultures (Fig. 5AGo). In addition, we analyzed the effect of CNP signaling on the expression of both Sox5 and Sox6 and demonstrated no significant difference (Fig. 5Go, B and C). To analyze the effects of CNP on Sox9 activity, we transfected cells with a Sox9-responsive luciferase reporter vector before micromass culture and stimulated the cells with 1 µM CNP or vehicle control. However, CNP treatment did not result in significant changes in luciferase activity (Fig. 5DGo). These data suggest that stimulation of chondrogenesis by CNP occurs independently of changes in Sox9 expression or Sox9 activity.


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 5. CNP signaling promotes chondrogenesis in a Sox9-independent manner A–C, RNA was isolated from micromass cultures treated with BSA/HCl control or 1 µM CNP for 6 d, and Sox9 (A), Sox5 (B), or Sox6 (C) mRNA levels were determined by real-time PCR. mRNA levels for all three genes did not change in response to CNP treatment in all time points studied. D, Micromass cultures were transfected with a firefly luciferase-based Sox9 reporter plasmid and pRlSV40 (encoding the Renilla luciferase gene under control of the SV40 promoter to standardize for transfection efficiency), plated, and cultured for 3 d with the vehicle or 1 µM CNP. Cells were harvested, and firefly luciferase activity was measured using the dual luciferase assay system (Promega) and standardized to Renilla luciferase activity. CNP treatment does not affect Sox9 activity. All data are the average relative gene expression ± SEM of at least three independent experiments. *, P < 0.05.

 
CNP signaling stimulates link but not collagen II or aggrecan
Although Sox9 activity did not change in response to treatment of micromass cultures with CNP, we analyzed expression of collagen II, aggrecan, and link transcripts. Expression of all three mRNAs increased over the culture period in control cultures. Collagen II and aggrecan mRNA levels demonstrated no change in expression in response to CNP (Fig. 6Go, A and B). However, analysis of link mRNA levels revealed a significant, almost 2-fold increase in mRNA levels in response to CNP by d 6 of culture (Fig. 6CGo).


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 6. Some components of the cartilage matrix are up-regulated in response to CNP signaling. A, Collagen II mRNA levels were quantified by real-time PCR and demonstrate no significant difference in comparison of control cultures with cultures treated with 1 µM CNP; B, real-time PCR demonstrated no significant changes in mRNA levels of aggrecan in response to CNP treatment; C, real-time PCR of link mRNA levels demonstrated a significant increased in micromass cultures treated with 1 µM CNP compared with control by d 6 of culture. Data shown are the average of three independent trials, relative gene expression ± SEM. *, P < 0.05.

 
We wanted to confirm that collagen II and aggrecan do not increase by analyzing protein expression. Immunohistochemistry of micromass cultures demonstrated that expression of both proteins increases during culture, but there is no visible increase in collagen II or aggrecan protein levels in response to CNP (Fig. 7Go, A and B). For both proteins, staining was observed within the nodules. The number of positively stained nodules for both collagen II and aggrecan were also counted from d-6 cultures and then normalized to total protein (Fig. 7Go, C and D), revealing no significant differences between control cultures and cultures treated with CNP.


Figure 7
View larger version (78K):
[in this window]
[in a new window]

  		FIG. 7. CNP treatment does not increase protein levels of collagen II or aggrecan. A and B, Micromass cultures treated with vehicle or 1 µM CNP were fixed and incubated with an antibody directed to collagen II (A) or aggrecan (B); C and D, the number of positive nodules for collagen II (C) and aggrecan (D) were counted and normalized to total protein. No visible or quantifiable differences exist for protein localization or the number of positive nodules in cultures treated with the vehicle or with CNP. Data shown are representative of three independent trials (A and B) and the average of three independent trials, normalized mean ± SEM (C and D).

 
Gene expression of matrix biosynthesis proteins are increased with CNP signaling
The matrix biosynthesis proteins xylosyltransferases I and II are necessary for the synthesis of chondroitin sulfate. mRNA levels for xylosyltransferase I increased strongly during chondrogenesis (Fig. 8AGo). CNP treatment resulted in a significant increase in expression of xylosyltransferase I. but not Xylosyltransferase II by d 6 of micromass culture (Fig. 8Go, A and B). Because proper sulfation of GAGs is also required for their function and because we had observed an increase in Alcian blue stain (suggesting an increase in sulfation), we analyzed expression of the chondroitin 6-sulfotransferase 3 (Chst3) or chondroitin 4-sulfotransferase 11 (Chst11) genes. CNP signaling resulted in a significant increase in mRNA levels of both sulfotransferases by d 6 of culture (Fig. 8Go, C and D).


Figure 8
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 8. CNP stimulates expression of genes involved in GAG synthesis. A, Real-time PCR demonstrated a significant increase of xylosyltransferase I mRNA with time as cultures become more chondrogenic. Furthermore, levels of xylosyltransferase I mRNA significantly increased in response to CNP treatment by d 3 of micromass culture compared with control. B, Xylosyltransferase II expression significantly increased over time in culture, but CNP treatment did not significantly change mRNA levels in comparison with control cultures. C, mRNA levels of the chondroitin 6-sulfotransferase 3 (Chst3) increases with time in micromass culture, and CNP treatment results in significant increase by d 6 of micromass culture. D, Chondroitin 4-sulfotransferase 11 (Chst11) mRNA levels increase with time in micromass culture, and by d 6 of micromass culture, CNP signaling increases mRNA levels. Data shown are an average of three independent trials, relative gene expression ± SEM. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CNP has been documented to regulate endochondral ossification through the promotion of chondrocyte proliferation and hypertrophy in the growth plate. We are the first to demonstrate a role for CNP in the early stages of chondrogenesis. We show that as cells become chondrogenic, they dramatically increase expression of the CNP receptor Npr2, providing a mechanism for CNP signaling. Compared with the strong increase in Npr2 levels, the biological effects of CNP treatment appear somewhat modest. One potential explanation is that the simultaneous up-regulation of Npr3, encoding the decoy receptor, limits cellular responses to CNP. These data suggest that the biological activities of CNP in cartilage are tightly regulated, in agreement with our recent observation that CNP induces Npr3 expression at later stages of chondrocyte differentiation (11).

We demonstrate that CNP treatment stimulates GAG accumulation. Furthermore, CNP treatment stimulates cellular condensations as demonstrated by increased PNA staining and mRNA levels of the cellular adhesion molecule N-cadherin. It is interesting that in response to CNP treatment, micromass cultures display a 60% increase in Alcian blue staining (after normalization to protein levels) but only a 30% increase in the number of condensations. These data suggest that the increase in Alcian blue staining is in part due to increased synthesis of GAGs per condensation and not just to an increase in the number of condensations.

Although these data suggest a promotion of chondrogenesis by CNP, we did not observe any changes in the expression or activity of chondrogenic Sox family members or changes in expression of the matrix molecules collagen II and aggrecan at the mRNA or protein level. However, transcripts for link protein were increased in response to CNP treatment, suggesting some effect of CNP on the formation of the proteoglycan aggregates. Interestingly, although the number of Alcian blue- and PNA-stained nodules increased in response to CNP treatment, the number collagen II- or aggrecan-stained nodules was not affected. This suggests that CNP preferentially stimulates condensations and GAG synthesis without parallel effects on other aspects of chondrogenesis.

Our data suggest that some aspects of chondrogenesis, for example GAG production, are at least partially independent of Sox9 activity. To assess the molecular mechanisms underlying increased GAG synthesis, we analyzed the expression levels of genes involved in GAG synthesis. Xylosyltransferase I expression increased strongly during chondrogenesis. In addition, CNP further increased levels of xylosyltransferase I but had no significant effects on the mRNA levels of xylosyltransferase II. We also demonstrate that both chondroitin 6-sulfotransferase 3 and chondroitin 4-sulfotransferase 11 are up-regulated in response to CNP signaling. These data suggest that the observed increase in Alcian blue stain is likely due to an increase in the synthesis and sulfation of GAG side chains through the up-regulation of xylosyltransferase I and sulfotransferases.

The identification of signaling pathways regulating chondrogenesis is essential for the development of novel therapeutics for diseases affecting cartilage. Not only have we demonstrated a novel role of CNP in the early stages of chondrogenesis, but we have also identified an important regulator of GAG synthesis. Although GAGs are an accepted marker of chondrogenesis, not much attention has been placed on its importance in regulating chondrocyte differentiation. Knockout animal models of enzymes regulating this biosynthetic pathway clearly demonstrate the importance of GAGs and their synthesis in the cartilage matrix (47, 52, 53). Not only can changes in GAG content interfere with structural and mechanical properties of the cartilage ECM, but they might also impede on the function of numerous growth factors dependent on interaction with proteoglycans and GAGs (54, 55, 56), as demonstrated, for example, for members of the TGF-ß family (47).

In conclusion, our data demonstrate that CNP signaling regulates chondrocyte differentiation at the stage of chondrogenesis, both through the regulation of cellular condensations and through the synthesis of GAGs. It is possible that these novel functions of CNP contribute both to growth retardation in response to loss of CNP signaling and to the potential therapeutic effects of exogenous CNP.


   Footnotes
 
A.W. was supported by graduate student awards from the Canadian Arthritis Network (CAN) and the Canadian Institutes of Health Research (CIHR), S.K. was also supported by a graduate student award from CAN, and F.B. is the recipient of a Canada Research Chair. Operating funds for these studies were provided by the CIHR and The Arthritis Society.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: CNP, C-type natriuretic peptide; ECM, extracellular matrix; GAG, glycosaminoglycan; Npr2, natriuretic peptide receptor 2; PNA, peanut agglutinin.

Received May 23, 2007.

Accepted for publication July 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosenzweig A, Seidman CE 1991 Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 60:229–255[CrossRef][Medline]
  2. Hagiwara H, Sakaguchi H, Itakura M, Yoshimoto T, Furuya M, Tanaka S, Hirose S 1994 Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B. J Biol Chem 269:10729–10733[Abstract/Free Full Text]
  3. Suda M, Ogawa Y, Tanaka K, Yasoda A, Takigawa T, Uehira M, Nishimoto H, Itoh H, Saito Y, Shiota K, Nakao K 1998 Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide. Proc Natl Acad Sci USA 95:2337–2342[Abstract/Free Full Text]
  4. Tamura N, Garbers DL 2003 Regulation of the guanylyl cyclase-B receptor by alternative splicing. J Biol Chem 278:48880–48889[Abstract/Free Full Text]
  5. Komatsu Y, Chusho H, Tamura N, Yasoda A, Miyazawa T, Suda M, Miura M, Ogaway Y, Nakao K 2002 Significance of C-type natriuretic peptide (CNP) in endochondral ossification: analysis of CNP knockout mice. J Bone Miner Res 20:331–336[CrossRef]
  6. Chusho H, Tamura N, Ogawa Y, Yasoda A, Suda M, Miyazawa T, Nakamura K, Nakao K, Kurihara T, Komatsu Y, Itoh H, Tanaka K, Saito Y, Katsuki M, Nakao K 2001 Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc Natl Acad Sci USA 98:4016–4021[Abstract/Free Full Text]
  7. Bartels CF, Bukulmez H, Padayatii P, Rhee DK, van Ravenswaij-Arts C, Pauli RM, Mundlos S, Chitayat D, Shih LY, Al-Gazali LI, Kant S, Cole T, Morton J, Cormier-Daire V, Faivre L, Kirk J, Mortier GR, Leroy J, Zabel B, Kim CA, Crow Y, Braveman NE, van den Akker F, Warman ML 2004 Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maroteaux. Am J Hum Genet 75:27–34[CrossRef][Medline]
  8. Olney RC, Bukulmez H, Bartels CF, Prickett TC, Espiner EA, Potter LR, Warman ML 2006 Heterozygous mutations in natriuretic peptide-B (NPR2) are associated with short stature. J Clin Endocrinal Metab 91:1229–1232[Abstract/Free Full Text]
  9. Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, Kurihara T, Rogi T, Tanaka S, Suda M, Tamura N, Ogawa Y, Nakao K 2004 Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med 10:80–86[CrossRef][Medline]
  10. Potter LR, Abbey-Hosch S, Dickey DM 2006 Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signalling functions. Endocr Rev 27:47–72[Abstract/Free Full Text]
  11. Agoston H, Khan S, James CG, Gillespie JR, Serra R, Stanton LA, Beier F 2007 C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways. BMC Dev Biol 7:18[CrossRef][Medline]
  12. Mericq V, Uyeda JA, Barnes KM, De Luca F, Baron J 2000 Regulation of fetal rat bone growth by C-type natriuretic peptide and cGMP. Pediatr Res 47:189–193[Medline]
  13. Fuishige K, Kotera J, Yanaka N, Akatsuka H, Omori K 1999 Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5. Biochim Biophys Acta 1452:219–227[Medline]
  14. Kronenberg HM 2003 Developmental regulation of the growth plate. Nature 423:332–336[CrossRef][Medline]
  15. Hall BK, Miyake T 2000 All for one and one for all: Condensations and the initiation of skeletal development. Bioessays 22:138–147[CrossRef][Medline]
  16. Ahrens PB, Solursh M, Reiters R 1977 Stage-related capacity for limb chondrogenesis in cell culture. Dev Biol 60:69–82[CrossRef][Medline]
  17. Erlebacher A, Filvaroff EH, Gitelman SE, Derynck R 1995 Toward a molecular understanding of skeletal development. Cell 80:371–378[CrossRef][Medline]
  18. Oberlender SA, Tuan RS 1994 Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 120:177–187[Abstract]
  19. DeLise AM, Tuan RS 2002 Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro. Dev Dynam 225:195–204[CrossRef][Medline]
  20. Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P 1994 N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp Cell Res 215:354–362[CrossRef][Medline]
  21. Fang J, Hall BK 1999 N-CAM is not required for initiation of secondary chondrogenesis: the role of N-CAM in skeletal condensation and differentiation. Int J Dev Biol 43:335–342[Medline]
  22. Tuan RS 2004 Biology of developmental and regenerative skeletogenesis. Clin Orthop Relat Res 427:S105–S117
  23. Muir H 1995 The chondrocyte, architecture of cartilage, biomechanics, structure, function and molecular biology of cartilage matrix macromolecules. Bioessays 17:1039–1048[CrossRef][Medline]
  24. Mwale F, Tchetina E, Wu CW, Poole AR 2002 The assembly and remodeling of the extracellular matrix in the growth plate in relationship to mineral deposition and cellular hypertrophy: an in situ study of collagens II and IX and proteoglycan. J Bone Miner Res 2:275–283
  25. Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B 2001 Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci USA 98:6698–6703[Abstract/Free Full Text]
  26. Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B 1999 Sox9 is required for cartilage formation. Nat Genet 22:85–89[CrossRef][Medline]
  27. Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling KW, Sham MW, Koopman P, Tam PP, Cheah KS 1997 SOX9 directly regulates the type-ll collagen gene. Nat Genet 16:174–178[CrossRef][Medline]
  28. Sekiya I, Tsuji K, Koopman P, Wantanabe H, Yamada Y, Shinomiya K, Nifuji A, Noda M 2000 Sox9 enhances aggrecan promoter/enhancer activity and is upregulated by retinoic acid in a cartilage derived cell-line TC6. J Biol Chem 275:10738–10744[Abstract/Free Full Text]
  29. Lefebvre V, Li P, de Crombrugghe B 1998 A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J 17:5718–5733[CrossRef][Medline]
  30. Doege KJ 1999 Aggrecan. In: Kreis T, Vale R, eds. Guidebook to the extracellular matrix, anchor and adhesion proteins. 2nd ed. Oxford, UK: Oxford University Press (a Sambrook and Tooze Publication); 359–361
  31. Bruckner P, van der Rest M 1994 Structure and function of cartilage collagens. Microsc Res Tech 28:378–384[CrossRef][Medline]
  32. Maleski MP, Knudson CB 1996 Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis. Exp Cell Res 225:55–66[CrossRef][Medline]
  33. Kimura JH, Hardingham TE, Hascall VC 1980 Assembly of newly synthesized proteoglycan and link protein into aggregates in cultures of chondrosarcoma chondrocytes. J Biol Chem 255:7134–7143[Abstract/Free Full Text]
  34. Watanabe H, Yamada Y, Kimata K 1998 Roles of aggrecan, a large chondroitin sulfate proteoglycan, in cartilage structure and function. J Biochem 124:687–693[Abstract/Free Full Text]
  35. Terry DE, Chopra RK, Ovenden J, Anastassiades TP 2000 Differential uses of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes. Anal Biochem 285:211–219[CrossRef][Medline]
  36. Krueger RC, Fields TA, Hildreth J, Schwartz NB 1990 Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment. J Biol Chem 265:12075–12087[Abstract/Free Full Text]
  37. Iozzo RV 1998 Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67:609–652[CrossRef][Medline]
  38. Keiser H, Shulman HJ, Sandson JI 1972 Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies. Biochem J 126:163–169[Medline]
  39. Kearns AE, Vertel BM, Schwartz NB 1993 Topography of glycosylation and UDP-xylose production. J Biol Chem 268:11097–11104[Abstract/Free Full Text]
  40. Woods A, Wang G, Dupuis H, Shao Z, Beier F 14 June 2007 Rac1 signaling stimulates N-cadherin expression, mesenchymal condensation and chondrogenesis. J Biol Chem 10.1074/jbc.M700680200
  41. Cuellar K, Chuong H, Hubbell SM, Hinsdale ME 2006 Biosynthesis of chondroitin and heparan sulfate in CHO cells depends on xylosyltransferase II. J Biol Chem 282:5195–5200[CrossRef][Medline]
  42. Prante C, Bieback K, Funke C, Schon S, Kern S, Kuhn J, Gastens M, Kleesiek K, Gotting C 2006 The formation of extracellular matrix during chondrogenic differentiation of mesenchymal stem cells correlates with increased levels of xylosyltransferase I. Stem Cells 24:2252–2261[Abstract/Free Full Text]
  43. Habuchi O 2000 Diversity and functions of glycosaminoglycan sulfotransferases. Biochim Biophys Acta 1474:115–127[Medline]
  44. Sugahara K, Kitagawa H 2000 Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr Opin Cell Biol 10:518–527
  45. Herndon ME, Lander AD 1990 A diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system. Neuron 4:949–961[CrossRef][Medline]
  46. Theile H, Sakano M, Kitagawa H, Sugahara K, Rajab A, Hohne W, Ritter H, Leschik G, Nurnberg P, Mundlos S 2004 Loss of chondroitin 6-O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement. Proc Natl Acad Sci USA 101:10155–10160[Abstract/Free Full Text]
  47. Kluppel M, Wight TN, Chan C, Hinek A, Wrana JL 2005 Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis. Development 132:3989–4003[Abstract/Free Full Text]
  48. Stanton LA, Sabari S, Sampaio AV, Underhill TM, Beier F 2004 p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation. Biochem J 378:53–62[CrossRef][Medline]
  49. Woods A, Wang G, Beier F 2005 RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. J Biol Chem 280:11626–11634[Abstract/Free Full Text]
  50. Weston AD, Chandraratna RAS, Torchia J, Underhill TM 2002 Requirement for RAR-mediated gene repression in skeletal progenitor differentiation. J Cell Biol 158:39–51[Abstract/Free Full Text]
  51. Lefebvre V, Smits P 2005 Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today 75:200–212[CrossRef][Medline]
  52. Rossi A, Superti-Furga A 2001 Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene (SLC26A2): 22 novel mutations, mutation review, associated skeletal phenotypes, and diagnostic relevance. Hum Mutat 17:159–171[CrossRef][Medline]
  53. Hastbacka J, Superti-Furga A, Wilcox WR, Rimoin DL, Cohn DH, Lander ES 1996 Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias. Am J Hum Genet 58:255–262[Medline]
  54. Koziel L, Kunath M, Kelly OG, Vortkamp A 2004 Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Developmental Cell 6:801–813[CrossRef][Medline]
  55. Crown SE, Sweeney MD, Leary JA, Handel TM 2006 Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding. J Biol Chem 281:25438–25446[Abstract/Free Full Text]
  56. Osses N, Gutierrez J, Lopez-Rovira T, Ventura F, Brandan E 2006 Sulfation is required for bone morphogenetic protein 2-dependent ID1 induction. Biochem Biophys Res Commun 344:1207–1215[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/10/5030    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woods, A.
Right arrow Articles by Beier, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woods, A.
Right arrow Articles by Beier, F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals