C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and – independent pathways

  • Hanga Agoston1,

    Affiliated with

    • Sameena Khan1,

      Affiliated with

      • Claudine G James1,

        Affiliated with

        • J Ryan Gillespie1,

          Affiliated with

          • Rosa Serra2,

            Affiliated with

            • Lee-Anne Stanton1 and

              Affiliated with

              • Frank Beier1Email author

                Affiliated with

                BMC Developmental Biology20077:18

                DOI: 10.1186/1471-213X-7-18

                Received: 04 August 2006

                Accepted: 20 March 2007

                Published: 20 March 2007

                Abstract

                Background

                C-type natriuretic peptide (CNP) has recently been identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanisms mediating its effects are not completely understood.

                Results

                We demonstrate in a tibia organ culture system that pharmacological inhibition of p38 blocks the anabolic effects of CNP. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We also performed Affymetrix microarray analyses on micro-dissected tibiae to identify CNP target genes. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. In addition, our data show that CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. Finally, detailed analyses of our microarray data showed that CNP regulates numerous genes involved in BMP signaling and cell adhesion.

                Conclusion

                Our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral bone growth, with potential implications for understanding and treatment of numerous skeletal diseases.

                Background

                Bone formation occurs through the related, but distinct processes of intramembranous and endochondral ossification [1, 2]. While the former is responsible for the formation of bones directly from precursor cells, such as the majority of the skull, the latter is responsible for the development of long bones, ribs, and vertebrae through a cartilage intermediate. In endochondral ossification, mesenchymal cells condense and begin to differentiate into chondrocytes, some of which later form the growth plate that controls longitudinal growth of endochondral bones [3]. The growth plate zones consists of resting, proliferating, and terminally differentiated hypertrophic chondrocytes, each of which are characterized by the expression of specific markers [4, 5]. This organization of the growth plate and the coordinated proliferation and hypertrophy of chondrocytes are responsible for elongation of bones and eventually determine final bone length. Hypertrophic chondrocytes are thought to undergo apoptosis, and simultaneously their surrounding cartilaginous matrix is degraded and replaced by bony tissue, produced by cells entering through vascularization of hypertrophic cartilage. The intricate control mechanisms regulating the proliferation, differentiation and apoptosis of chondrocytes as well as the subsequent vascular invasion are not completely understood. However, disturbances of these processes can result in numerous diseases such as chondrodysplasias and other growth disorders, demonstrating the need for a better understanding of the pathways involved [4, 69].

                C-type natriuretic peptide (CNP) has recently been shown to be an important regulator of endochondral ossification. The dominant phenotype of CNP-deficient mice is dwarfism, as demonstrated by shortened long bones primarily due to reduced heights of proliferating and hypertrophic zones of the growth plate [10, 11]. CNP also increases growth in mouse bone organ cultures [12, 13]. More recently, loss-of-function mutations in NPR2, the gene encoding the CNP receptor, have been identified as cause of acromesomelic dysplasia type Maroteaux, an autosomal recessive chondrodysplasia in humans [14]. This CNP receptor is also known as guanylyl cyclase-B (GC-B) or NPR-B. Binding of CNP to GC-B results in increased intracellular cGMP, which can further activate downstream factors, such as cGKI and cGKII (cGMP-dependent protein kinase I and II) as well as phosphodiesterases (PDEs) that break down cGMP and camp and specific ion channels [1518]. In addition, CNP can also bind to a different receptor, NPR-3 (natriuretic peptide receptor 3) that is thought to act as a decoy/clearance receptor serving to limit the effects of natriuretic peptides. Interestingly, mice deficient for cGKII show a similar, although not identical phenotype to CNP-deficient mice [19], and cGKII has been shown to be required for the effects of CNP overexpression in transgenic mice [20]. These data clearly identify cGKII as an essential mediator of CNP effects, but the signaling pathways downstream of cGKII, potential parallel pathways and the target genes conferring cartilage responses to CNP are not completely understood. However, recent studies showed that overexpression of CNP results in inhibition of the MEK1/2-ERK1/2 MAP kinase pathway and rescues the effects of an activating fibroblast growth factor receptor 3 mutation on endochondral bone growth [2123].

                MAP kinases are central signaling molecules in most eukaryotic cells that integrate extracellular signals leading to altered cell proliferation, differentiation, and transcription in many cell types, including chondrocytes [24, 25]. For example, both the ERK and the p38 MAPK families have been shown to play important roles in controlling chondrocyte differentiation in vitro and in vivo [3, 26, 27]. In the current study we demonstrate, for the first time, an essential role for the p38 MAP kinase pathway in CNP signaling in cartilage and identify target genes of CNP in chondrocytes using genome-wide microarrays.

                Results

                CNP signaling enhances endochondral bone growth

                We used an organ culture system of embryonic day 15.5 (E15.5) mouse tibiae to examine the effects of CNP on endochondral bone growth. Tibiae were cultured for six days in the presence of BSA (control) or different concentrations of CNP. 10-8, 10-7 and 10-6M concentrations of CNP caused a 31%, 40%, and 42% increase, respectively, in longitudinal growth of tibiae (Fig. 1A,B). Treatment with 1 μM CNP almost doubled tibia weight relative to controls (Fig. 1C). Incubation of tibiae with 10-4 M (8-(4-chlorophenylthio) cGMP stimulated tibia growth in a similar or stronger manner (55%) as CNP (Fig. 1D). A general inhibitor of phosphodiesterases (PDEs), 3-Isobutyl-1-methylxanthine (IBMX) at 10-4 M, was used to study the role of PDEs in bone growth in the organ cultures. PDE inhibition stimulated longitudinal growth by 30% when compared to the control. In contrast, specific inhibition of PDE 1 by 8-methoxymethyl IBMX did not alter bone growth significantly, indicating that this enzyme is either not involved in regulating bone growth or can be functionally replaced by other proteins. These data demonstrate that CNP/cGMP signaling stimulates endochondral bone growth, while PDEs inhibit this process. Removal of the perichondrium by enzymatic digestion and/or manual dissection did not alter the response to CNP, demonstrating that the anabolic effects of CNP are independent of the perichondrium (Fig. 1E).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig1_HTML.jpg
                Figure 1

                CNP enhances endochondral bone growth. Mouse E15.5 tibiae were harvested and cultured for six days in the presence of vehicle, CNP at the indicated concentrations, membrane-permeable 8-(4-cpt) cGMP (0.1 mM), the non-specific PDE inhibitor IBMX (0.1 mM), or a selective inhibitor of PDE I, 8-methooxymethyl, IBMX (10 μM). After six days in culture, vehicle and CNP-treated (1 μM) bones were stained with Alcian Blue and Alizarin Red and representative images are shown, in comparison to a freshly isolated tibia (A). Growth of tibiae over the culture period at indicated concentrations of CNP and treatments was measured (B, D), and the weight of bones was determined (C). CNP, 8-(4-cpt) cGMP and IBMX stimulated tibia growth, when compared to control conditions. E15.5 tibiae were isolated under three different conditions: perichondrium was left intact with very loose dissection, perichondrium was removed with dispase, and perichondrium was removed mechanically (E). Bones were then incubated with or without CNP (1 μM) for six days and bone growth was determined as change in bone length relative to day 1. Removal of the perichondrium did not influence the stimulatory effect of CNP on bone growth. All data represent means ± SD of three or four independent trials (p < 0.05).

                At the histological level, the most significant effect of CNP was a marked expansion of the hypertrophic zone (Fig. 2A). This enlargement of the hypertrophic zone was accomplished by increases in both the number and maximal size of hypertrophic chondrocytes (Fig. 2B), in agreement with earlier studies [23].
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig2_HTML.jpg
                Figure 2

                CNP induces expansion of the hypertrophic zone. Hematoxylin and Eosin staining of tibia sections after six days of culture with or without CNP (1 μM) showed differences in growth plate architecture, primarily in the hypertrophic zone. CNP treatment results in a vastly expanded hypertrophic zone (A; hypertrophic zones indicated by brackets). Magnification of cells in the hypertrophic zone (boxes from A) shows that individual chondrocytes are larger in CNP-treated tibiae (B).

                CNP-induced endochondral bone growth requires p38 MAP kinase signaling

                MAP kinases play multiple roles in chondrocyte differentiation and cartilage development [3]. We therefore examined a potential role of the MEK (MAP/ERK kinase) 1/2-ERK1/2 and p38 cascades in CNP-induced endochondral bone growth. In the absence of exogenous CNP, the pharmacological MEK1/2 inhibitors PD98059 and U0126 (10 μM each) stimulated tibia growth by 39% and 30%, respectively (Fig. 3A). While simultaneous addition of MEK inhibitors and CNP had maximal effects on bone growth, these effects were not statistically different from treatment with CNP alone. These data suggest that CNP and MEK1/2 act through a common pathway and are in agreement with recent studies demonstrating an inhibitory role of the MEK/ERK cascade in endochondral bone growth [26] and down-regulation of MEK/ERK activity by CNP [2123].
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig3_HTML.jpg
                Figure 3

                Inhibition of the MEK1/2-ERK1/2 pathway stimulates tibia growth, while p38 MAPK is required for CNP-induced bone growth. Mouse E15.5 tibiae were harvested and cultured for six days in the presence of control or CNP (1 μM) and vehicle (DMSO) or MEK1/2-ERK1/2 pathway inhibitors PD98059 (10 μM) and U0126 (10 μM) (A). Though both PD98059 and U0126 stimulated basal bone growth, inhibition of the MEK1/2-ERK1/2 pathway did not further enhance CNP-induced bone growth (*: p < 0.05 when comparing control/inhibitors to control/vehicle; #: p < 0.05 when comparing CNP/vehicle to control/vehicle; p > 0.05 when comparing CNP/vehicle to CNP/inhibitors). Tibiae were incubated with control or CNP and pharmacological inhibitors of the p38 MAPK pathway (SB202190 or PD169316, 10 μM each) or an inactive analog (SB202474, 10 μM) (B). p38 inhibition did not effect basal bone growth significantly, but did suppress CNP-induced bone growth (*: p < 0.05 when comparing CNP/inhibitors to CNP/SB202474; #: p < 0.05 when comparing CNP/SB202474 to control/SB202474). Bone growth was measured over an extended time course of eight days, showing that CNP continued to significantly influence growth on day 8, while SB202190 reversed these effects (C). Bones from each treatment were weighed under different conditions, and it was found that p38 inhibition reversed the effects of CNP on weight (D). Protein extracts from primary chondrocytes cultured with control, CNP (10-6M), or 8-(4-cpt) cGMP (0.1 mM) for 10 minutes were examined for phosphorylation of the p38 activators MKK3/6 by western blot analysis (E). Both treatments increased phosphorylation of MKK3/6, supporting the stimulation of p38 MAP kinase activity by CNP signaling. Immunohistochemistry with an antibody against phosphorylated p38 demonstrates markedly higher signal in CNP-treated tibiae when compared to control bones (F).

                We next examined whether p38 is involved in the effects of CNP on cartilage growth. Inhibition of p38 activity by two different compounds, PD169316 or SB202190 (10 μM each), did not affect basal endochondral bone growth, when compared to the inactive control compound SB202474 (10 μM) (Fig. 3B). In contrast, inhibition of p38 by SB202190 or PD169316 blocked CNP-induced growth (Fig. 3B). This effect was obvious by day 6 of culture and maintained by day 8 (Fig. 3C). Moreover, p38 inhibition completely reversed CNP effects on tibia weight (Fig. 3D), further demonstrating a requirement for p38 activity in CNP-induced endochondral bone growth. Next we examined whether CNP regulates the p38 pathway by investigating the phosphorylation of the kinases MKK3 (MAP kinase kinase3) and MKK6, direct and specific activators of p38. Western blotting with phospho-specific antibodies revealed that CNP and cGMP increase the phosphorylation of MKK3/6 in primary chondrocytes after 10 minutes of incubation (Fig. 3E), demonstrating that CNP signaling activates the p38 pathway in chondrocytes. Immunohistochemistry for phosphorylated (active) p38 showed little staining under control conditions, but demosntrated a strong increase in p38 phosphorylation in CNP-treated tibiae (Fig. 3F).

                CNP delays tibia mineralization in a p38-dependent manner

                To examine the effects of p38 inhibition on growth plate organization, we performed histological analyses of organ culture sections. As above, CNP stimulation caused an expansion of the hypertrophic zone of the growth plate, while SB202190 by itself did not have any marked effects (Fig. 4A). However, p38 inhibition suppressed the enlargement of the hypertrophic zone in response to CNP, providing further evidence for a requirement for p38 activity for the anabolic effects of CNP.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig4_HTML.jpg
                Figure 4

                p38 MAPK activity is required for CNP-induced hypertrophy. E15.5 tibiae were isolated and incubated with or without CNP (1 μM) and DMSO or SB202190 (10 μM). Hematoxylin and Eosin staining of tibia sections after six days of culture show that p38 inhibition reversed CNP-induced expansion of the hypertrophic zone (A). Tibiae were stained with Alizarin Red and Alcian Blue, and representative images demonstrate increased bone growth by CNP and the reversal of these effects upon p38 inhibition (B). The area of the mineralized zone (red) was measured as absolute area (C, bottom) and as a percentage of total area (C, top), demonstrating that CNP-treated bones displayed significantly smaller mineralized area in relation to the whole bone area. This was reversed upon p38 inhibition. Representative images are shown, while all data represent means ± SD of four independent trials, each with six bones (p < 0.05).

                During dissections and analyses of organ cultures, we also noticed that CNP-treated bones were more fragile and appeared less mineralized. Alcian Blue/Alizarin Red staining of tibiae confirmed that the mineralized area was smaller in CNP-treated bones and displayed weaker Alizarin Red staining (Fig. 4B). We quantified the area of the mineralized (red) and cartilaginous (blue) regions of tibiae using digital image analyses. CNP treatment did increase the Alcian Blue-stained area considerably, without effects on the Alizarin Red-stained area (Fig. 4C). This resulted in a reduction of the mineralized area relative to the total area of the bone by about 30%. These data suggests that CNP-induced growth of cartilage is not matched by a corresponding expansion of the mineralized area and that CNP treatment delays the remodeling of hypertrophic cartilage. Inhibition of p38 activity by SB202190 resulted in a slight, but significant increase of the mineralized area (relative to total area) and reversed the effects of CNP on the Alcian Blue-stained area completely (Fig. 4C).

                Microarray analyses identify hypertrophic chondrocytes as main targets of CNP signaling

                We next performed microarray analyses to identify target genes of CNP in chondrocytes. Tibiae were cultured for six days in the absence or presence of CNP and then micro-dissected into three distinct zones: the resting/proliferative (RP), the hypertrophic (H), and the mineralized (M) zones (Fig. 5A). RNA was isolated directly from tibiae from three independent trials for each zone and both treatments were analyzed using Affymetrix Mouse 2.0 arrays in the London Regional Genomics Center as described (London, Ontario, Canada) [28]. Real-time PCR analyses of collagen II (Col2a1) and collagen X (Col10a1), known markers of cartilage development, confirmed that micro-dissection resulted in efficient separation of the zones (Fig. 5B). Microarray profiles of selected genes involved in endochondral bone growth are shown to further illustrate correct separation of zones (Fig. 5C).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig5_HTML.jpg
                Figure 5

                Micro-dissection efficiently separates different growth plate zones from cultured tibiae. E15.5 tibiae that were harvested and incubated with or without CNP (1 μM) for six days were micro-dissected into the resting/proliferating, hypertrophic, and mineralized regions as shown (A). Zones from approximately 24 bones were pooled together. RNA was isolated directly from micro-dissected tibia and analyzed by microarray as described in Materials and Methods. Real-time PCR analyses confirmed expected expression patterns of the cartilage markers Col2a1 and Col10a1 in control bones (B; data represent means ± SD from three independent trials). Expression patterns of selected chondrocyte marker genes under control conditions in our microarray data sets further demonstrated efficient separation of regions (C).

                Bioinformatics analyses of microarray results (Fig. 6A) demonstrated that the hypertrophic zone was most responsive to CNP (Fig. 6B). Only 47 probe sets in the resting/proliferative zone (35 down, 12 up) and 58 probe sets in the mineralized zone (41 down, 17 up) responded with minimum two-fold responses to CNP (see Table 1 and 2 for lists of regulated genes). In contrast, 309 probe sets in the hypertrophic area showed a two-fold or higher change in expression in response to CNP. Of these probe sets, 157 probe sets were up-regulated by CNP in the hypertrophic zone, and 152 were down-regulated by CNP (Table 3).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig6_HTML.jpg
                Figure 6

                Microarray analyses identify the hypertrophic area as the main target of CNP treatment. E15.5 tibiae were isolated, incubated with or without CNP (1 μM) and DMSO or SB202190 (10 μM) and micro-dissected into the resting/proliferating, hypertrophic, and mineralized regions prior to RNA extraction and microarray analyses. Analyses of microarray results from three independent trials using Genespring 7.2 (A) illustrated that the hypertrophic zone was most significantly responsive to CNP treatment, when compared to control conditions (B). Six times as many probe sets showed at least 2-fold expression changes in the hypertrophic zone when compared to either resting/proliferating or mineralized regions. Real-time PCR analyses on micro-dissected tibiae were used to validate selected microarray patterns. CNP induction of Ptgs2, the gene encoding cyclooxygenase-2, was confirmed (C). SB202190 treatment did reduce basal Cox2 mRNA levels, but did not interfere with CNP induction of Cox2. Tnfsf11, the gene encoding RANKL, was confirmed to be down-regulated in response to CNP treatment. Data represent means ± SD of three independent trials (p < 0.05).

                Table 1

                Genes showing 2-fold or greater changes in resting/proliferating zone

                Gene Name

                Gene Description

                Fold Change (CNP/Control)

                Spock3

                sparc/osteonectin, cwcv and kazal-like domains proteoglycan 3

                2.76

                Il15

                interleukin 15

                2.73

                Mgst2

                microsomal glutathione S-transferase 2

                2.60

                Alcam

                activated leukocyte cell adhesion molecule

                2.52

                Tec

                cytoplasmic tyrosine kinase, Dscr28C related (Drosophila)

                2.27

                Aard

                alanine and arginine rich domain containing protein

                2.24

                Tnfsf11

                tumor necrosis factor (ligand) superfamily, member 11

                2.18

                Vegfc

                vascular endothelial growth factor C

                2.18

                Senp8

                SUMO/sentrin specific protease family member 8

                2.09

                Pde4b

                phosphodiesterase 4B, cAMP specific

                2.02

                Gcnt2

                glucosaminyl (N-acetyl) transferase 2, I-branching enzyme

                0.18

                Cidea

                cell death-inducing DNA fragmentation factor, alpha subunit-like

                0.24

                Frzb

                frizzled-related protein

                0.28

                F5

                coagulation factor V

                0.31

                Car8

                carbonic anhydrase 8

                0.32

                Ibsp

                integrin binding sialoprotein

                0.34

                Lifr

                leukemia inhibitory factor receptor

                0.36

                Col10a1

                procollagen, type X, alpha 1

                0.37

                Lepr

                leptin receptor

                0.37

                Ibsp

                integrin binding sialoprotein

                0.38

                Pthr1

                parathyroid hormone receptor 1

                0.38

                Clcn7

                chloride channel 7

                0.41

                Chd7

                chromodomain helicase DNA binding protein 7

                0.42

                H2-DMa

                histocompatibility 2, class II, locus DMa

                0.42

                Col13a1

                procollagen, type XIII, alpha 1

                0.42

                Cntn1

                contactin 1

                0.43

                Slc7a3

                solute carrier family 7, member 3

                0.44

                Tm7sf1

                transmembrane 7 superfamily member 1

                0.45

                Iqsec1

                IQ motif and Sec7 domain 1

                0.45

                Pthlh

                parathyroid hormone-like peptide

                0.46

                Pdzk3

                PDZ domain containing 2

                0.46

                Tmie

                transmembrane inner ear

                0.47

                Ifitm5

                interferon induced transmembrane protein 5

                0.47

                Socs2

                suppressor of cytokine signaling 2

                0.47

                Angptl2

                angiopoietin-like 2

                0.48

                Capn6

                calpain 6

                0.48

                Atp10d

                ATPase, Class V, type 10D

                0.48

                Cald1

                caldesmon 1

                0.49

                Phtf2

                putative homeodomain transcription factor 2

                0.50

                Table 2

                Genes showing 2-fold or greater changes in mineralized zone

                Gene Name

                Gene Description

                Fold Change (CNP/Control)

                Ptgs2

                prostaglandin-endoperoxide synthase 2

                2.87

                Bcan

                brevican

                2.75

                Gabrb3

                gamma-aminobutyric acid (GABA-A) receptor, subunit beta 3

                2.46

                Robo4

                roundabout homolog 4 (Drosophila)

                2.31

                Cd38

                CD38 antigen

                2.23

                Lepr

                leptin receptor

                2.22

                Cd24a

                CD24a antigen

                2.15

                Tgfbi

                transforming growth factor, beta induced

                2.14

                Tmem56

                transmembrane protein 56

                2.12

                Gsg2

                germ cell-specific gene 2

                2.11

                Dspg3

                dermatan sulphate proteoglycan 3

                2.11

                Siat4c

                sialyltransferase 4C (beta-galactoside alpha-2,3-sialytransferase)

                2.04

                Hbb-y

                hemoglobin Y, beta-like embryonic chain

                0.21

                Zbtb8

                zinc finger and BTB domain containing 8

                0.24

                Chic1

                cysteine-rich hydrophobic domain 1

                0.27

                Mia

                EGL nine homolog 2 (C. elegans)

                0.28

                Nox4

                NADPH oxidase 4

                0.29

                Fgd6

                FYVE, RhoGEF and PH domain containing 6

                0.32

                Ddc

                dopa decarboxylase

                0.34

                Crxos1

                Crx opposite strand transcript 1

                0.37

                Hapln1

                cartilage link protein 1

                0.37

                Col27a1

                procollagen, type XXVII, alpha 1

                0.38

                Fgd5

                FYVE, RhoGEF and PH domain containing 5

                0.38

                Msi2h

                Musashi homolog 2 (Drosophila)

                0.39

                Rgs11

                regulator of G-protein signaling 11

                0.42

                Mrpl35

                mitochondrial ribosomal protein L35

                0.43

                Wwp2

                WW domain containing E3 ubiquitin protein ligase 2

                0.44

                Glt25d2

                glycosyltransferase 25 domain containing 2

                0.44

                Zcchc5

                zinc finger, CCHC domain containing 5

                0.45

                Stno

                strawberry notch homolog (Drosophila)

                0.46

                Ppp1r3c

                protein phosphatase 1, regulatory (inhibitor) subunit 3C

                0.46

                Col9a3

                procollagen, type IX, alpha 3

                0.47

                Igf2

                insulin-like growth factor 2

                0.47

                Edil3

                EGF-like repeats and discordin I-like domains 3

                0.48

                Ttll3

                tubulin tyrosine ligase-like family, member 3

                0.48

                A2m

                alpha-2-macroglobulin

                0.49

                Ctf1

                cardiotrophin 1

                0.49

                Xist

                inactive X specific transcripts

                0.49

                Zfp458

                zinc finger protein 458

                0.50

                Table 3

                Genes showing 2-fold or greater changes in hypertrophic zone

                Gene Name

                Gene Description

                Fold Change (CNP/Control)

                Cxcl14

                chemokine (C-X-C motif) ligand 14

                7.15

                Ptgs2

                prostaglandin-endoperoxide synthase 2

                6.77

                Grem1

                cysteine knot superfamily 1, BMP antagonist 1

                6.47

                Fbxo32

                F-box only protein 32

                5.87

                Glipr1

                GLI pathogenesis-related 1 (glioma)

                5.22

                Gdf5

                growth differentiation factor 5

                4.93

                Nox4

                NADPH oxidase 4

                3.78

                Ebi2

                Epstein-Barr virus induced gene 2

                3.78

                Evi1

                ecotropic viral integration site 1

                3.50

                Prnd

                prion protein dublet

                3.43

                Acdc

                adipocyte complement related protein

                3.42

                Nes

                nestin

                3.33

                Tnnt3

                troponin T3, skeletal, fast

                3.29

                Nox4

                NADPH oxidase 4

                3.27

                Rbp1

                retinol binding protein 1, cellular

                3.19

                Hist1h2bc

                histone 1, H2bp

                3.16

                Inhbb

                inhibin beta-B

                3.12

                Sox17

                SRY-box containing gene 17

                3.10

                Rnf125

                ring finger protein 125

                3.08

                Fabp4

                fatty acid binding protein 4, adipocyte

                3.07

                C1ql3

                C1q-like 3

                3.03

                Rbpms

                RNA binding protein gene with multiple splicing

                3.02

                Nrarp

                Notch-regulated ankyrin repeat protein

                3.02

                Mmrn2

                multimerin 2

                3.00

                Cldn5

                claudin 5

                3.00

                Cd44

                CD44 antigen

                2.99

                Klhl4

                kelch-like 4 (Drosophila)

                2.98

                Pscd4

                pleckstrin homology, Sec7 and coiled/coil domains 4

                2.90

                Ptprc

                protein tyrosine phosphatase, receptor type, C

                2.86

                Rasgrp1

                RAS guanyl releasing protein 1

                2.85

                Ptger2

                prostaglandin E receptor 2 (subtype EP2)

                2.84

                Ctla2b

                trophoblast specific protein beta

                2.84

                Copg2as2

                coatomer protein complex, subunit gamma 2, antisense 2

                2.82

                Ian1

                immune associated nucleotide 1

                2.82

                Pmaip1

                phorbol-12-myristate-13-acetate-induced protein 1

                2.77

                Gpihbp1

                GPI-anchored HDL-binding protein 1

                2.73

                Cdh5

                cadherin 5

                2.69

                Niban

                niban protein

                2.67

                Ptpn3

                protein tyrosine phosphatase, non-receptor type 3

                2.67

                Slc26a7

                solute carrier family 26, member 7

                2.67

                Tm6sf1

                transmembrane 6 superfamily member 1

                2.65

                Pkp2

                plakophilin 2

                2.65

                Bcl2a1a

                B-cell leukemia/lymphoma 2 related protein A1a

                2.63

                Prss8

                protease, serine, 8 (prostasin)

                2.61

                Fads3

                fatty acid desaturase 3

                2.60

                Runx1

                runt related transcription factor 1

                2.56

                Abcc9

                ATP-binding cassette, sub-family C (CFTR/MRP), member 9

                2.56

                Nr2f1

                nuclear receptor subfamily 2, group F, member 1

                2.55

                Hbb-y

                hemoglobin Y, beta-like embryonic chain

                2.52

                Akr1b8

                aldo-keto reductase family 1, member B8

                2.51

                Siat8f

                sialyltransferase 8 (alpha-2, 8-sialyltransferase) F

                2.51

                Sfpi1

                SFFV proviral integration 1

                2.51

                Zbtb33

                zinc finger and BTB domain containing 33

                2.49

                Gdpd1

                glycerophosphodiester phosphodiesterase domain containing 1

                2.46

                Clecsf6

                C-type lectin, superfamily member 6

                2.46

                Pstpip1

                proline-serine-threonine phosphatase-interacting protein 1

                2.45

                Esam1

                endothelial cell-specific adhesion molecule

                2.44

                Cdh13

                cadherin 13

                2.43

                Hist2h3c2

                histone 2, H2aa1

                2.43

                Sfrp2

                secreted frizzled-related sequence protein 2

                2.40

                Cables1

                Cdk5 and Abl enzyme substrate 1

                2.39

                Ednrb

                endothelin receptor type B

                2.39

                Eltd1

                EGF, latrophilin seven transmembrane domain containing 1

                2.38

                Calcrl

                calcitonin receptor-like

                2.38

                Ctla2b

                trophoblast specific protein beta

                2.38

                Ian1

                immune associated nucleotide 1

                2.36

                Sox18

                SRY-box containing gene 18

                2.36

                Plce1

                phospholipase C, epsilon 1

                2.33

                Il13ra1

                interleukin 13 receptor, alpha 1

                2.33

                Cd38

                CD38 antigen

                2.32

                Ncf4

                neutrophil cytosolic factor 4

                2.30

                Rgs4

                regulator of G-protein signaling 4

                2.30

                Ptpn8

                protein tyrosine phosphatase, non-receptor type 8

                2.29

                Inhba

                inhibin beta-A

                2.29

                Alcam

                activated leukocyte cell adhesion molecule

                2.27

                Pira1

                paired-Ig-like receptor A1

                2.27

                Cav2

                caveolin 2

                2.27

                Cxcr4

                chemokine (C-X-C motif) receptor 4

                2.26

                Sh3bp5

                calpain 7

                2.26

                Mfap3l

                microfibrillar-associated protein 3-like

                2.26

                Dscr1

                Down syndrome critical region homolog 1 (human)

                2.25

                Mcam

                melanoma cell adhesion molecule

                2.24

                Ms4a6d

                membrane-spanning 4-domains, subfamily A, member 6D

                2.24

                Cd34

                CD34 antigen

                2.24

                Zfp42

                zinc finger protein 42

                2.23

                Kcne3

                potassium voltage-gated channel, Isk-related subfamily, gene 3

                2.22

                Ivns1abp

                influenza virus NS1A binding protein

                2.22

                Cd84

                CD84 antigen

                2.22

                Kdr

                kinase insert domain protein receptor

                2.21

                Clca5

                chloride channel calcium activated 5

                2.20

                Itga9

                integrin alpha 9

                2.19

                Prkch

                protein kinase C, eta

                2.19

                Tex15

                testis expressed gene 15

                2.18

                Plac8

                placenta-specific 8

                2.17

                Ebf3

                early B-cell factor 3

                2.16

                Lcp2

                lymphocyte cytosolic protein 2

                2.16

                Mcoln3

                mucolipin 3

                2.15

                Sh3glb1

                SH3-domain GRB2-like B1 (endophilin)

                2.15

                Ugt1a2

                UDP glycosyltransferase 1 family, polypeptide A6

                2.15

                Egfl7

                EGF-like domain 7

                2.15

                Icam2

                intercellular adhesion molecule 2

                2.15

                Six1

                sine oculis-related homeobox 1 homolog (Drosophila)

                2.14

                Chst7

                carbohydrate (N-acetylglucosamino) sulfotransferase 7

                2.13

                Evi2a

                ecotropic viral integration site 2a

                2.12

                Myct1

                myc target 1

                2.12

                Pde4b

                phosphodiesterase 4B, cAMP specific

                2.12

                Adamts1

                a disintegrin-like & metalloprotease with thrombospondin type 1

                2.10

                Snx10

                sorting nexin 10

                2.10

                Rac2

                RAS-related C3 botulinum substrate 2

                2.09

                Siat8d

                sialyltransferase 8 (alpha-2, 8-sialyltransferase) D

                2.08

                Dsg2

                desmoglein 2

                2.07

                F11r

                F11 receptor

                2.06

                Lrrc33

                leucine rich repeat containing 33

                2.06

                Ian9

                Similar to hypothetical protein (LOC243374), mRNA

                2.06

                Slc30a1

                solute carrier family 30 (zinc transporter), member 1

                2.05

                Kcnj8

                potassium inwardly-rectifying channel, subfamily J, member 8

                2.05

                Cotl1

                coactosin-like 1 (Dictyostelium)

                2.04

                Ptx3

                pentaxin related gene

                2.04

                Ctla2b

                trophoblast specific protein beta

                2.03

                Sipa1

                signal-induced proliferation associated gene 1

                2.03

                Rgs5

                regulator of G-protein signaling 5

                2.03

                Itgax

                integrin alpha X

                2.01

                Car2

                carbonic anhydrase 2

                2.01

                Serpind1

                serine (or cysteine) proteinase inhibitor, clade D, member 1

                2.01

                Cadps2

                Ca2+-dependent activator protein for secretion 2

                2.00

                Il1rl2

                interleukin 1 receptor-like 2

                2.00

                Lemd1

                LEM domain containing 1

                0.06

                Gzme

                granzyme E

                0.14

                Plekha7

                pleckstrin homology domain containing, family A member 7

                0.14

                Chad

                chondroadherin

                0.15

                Cd28

                CD28 antigen

                0.17

                Sep-04

                septin 4

                0.19

                Pltp

                phospholipid transfer protein

                0.19

                Il15

                interleukin 15

                0.20

                Ttll3

                tubulin tyrosine ligase-like family, member 3

                0.20

                Syt8

                synaptotagmin 8

                0.20

                Gpr91

                G protein-coupled receptor 91

                0.23

                Sep-04

                septin 4

                0.24

                Cd28

                CD28 antigen

                0.24

                Efemp1

                epidermal growth factor-containing fibulin-like ECM protein 1

                0.25

                Tnfsf11

                tumor necrosis factor (ligand) superfamily, member 11

                0.27

                Tlr1

                toll-like receptor 1

                0.28

                Trim2

                tripartite motif protein 2

                0.28

                Vnn1

                vanin 1

                0.28

                Tnni2

                troponin I, skeletal, fast 2

                0.29

                Enpp6

                ectonucleotide pyrophosphatase/phosphodiesterase 6

                0.30

                Fxyd2

                FXYD domain-containing ion transport regulator 2

                0.30

                Rtn2

                reticulon 2 (Z-band associated protein)

                0.31

                Iqgap2

                IQ motif containing GTPase activating protein 2

                0.31

                Capn6

                calpain 6

                0.32

                Rab27a

                RAB27A, member RAS oncogene family

                0.32

                Aicda

                activation-induced cytidine deaminase

                0.33

                F5

                coagulation factor V

                0.33

                Hs6st2

                heparan sulfate 6-O-sulfotransferase 2

                0.33

                Cklfsf8

                chemokine-like factor super family 8

                0.33

                Nrk

                Nik related kinase

                0.33

                Gprasp2

                G protein-coupled receptor associated sorting protein 2

                0.33

                Car8

                carbonic anhydrase 8

                0.34

                Prom1

                prominin 1

                0.34

                Mgst2

                microsomal glutathione S-transferase 2

                0.34

                Pltp

                phospholipid transfer protein

                0.35

                Stc2

                stanniocalcin 2

                0.35

                Lipg

                lipase, endothelial

                0.35

                Il17d

                interleukin 17D

                0.36

                Serpinb6b

                serine (or cysteine) proteinase inhibitor, clade B, member 6b

                0.36

                Matn3

                matrilin 3

                0.37

                Slc1a1

                solute carrier family 1, member 1

                0.37

                Art3

                ADP-ribosyltransferase 3

                0.37

                Cp

                ceruloplasmin

                0.37

                Abi3bp

                ABI gene family, member 3 (NESH) binding protein

                0.37

                Matn1

                matrilin 1, cartilage matrix protein 1

                0.38

                A2m

                alpha-2-macroglobulin

                0.38

                Usp11

                ubiquitin specific protease 11

                0.38

                Col9a2

                procollagen, type IX, alpha 2

                0.39

                Pik3r1

                phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1

                0.39

                Rlbp1

                retinaldehyde binding protein 1

                0.39

                Rnase4

                ribonuclease, RNase A family 4

                0.39

                Col14a1

                procollagen, type XIV, alpha 1

                0.40

                Slc19a3

                solute carrier family 19 (sodium/hydrogen exchanger), member 3

                0.40

                Nfkbiz

                NFK light polypeptide gene enhancer in B-cells inhibitor, zeta

                0.40

                Slco2b1

                solute carrier organic anion transporter family, member 2b1

                0.41

                Fxyd6

                FXYD domain-containing ion transport regulator 6

                0.41

                Egr3

                early growth response 3

                0.41

                Usp53

                ubiquitin specific peptidase 53

                0.41

                Serpini1

                serine (or cysteine) proteinase inhibitor, clade I, member 1

                0.41

                Pitpnc1

                phosphatidylinositol transfer protein, cytoplasmic 1

                0.42

                Anxa8

                annexin A8

                0.42

                Il17b

                interleukin 17B

                0.43

                Gpr126

                G protein-coupled receptor 126

                0.43

                Pank1

                pantothenate kinase 1

                0.43

                Dock9

                dedicator of cytokinesis 9

                0.43

                Sfmbt2

                Scm-like with four mbt domains 2

                0.43

                Enpp2

                ectonucleotide pyrophosphatase/phosphodiesterase 2

                0.44

                Kctd4

                potassium channel tetramerisation domain containing 4

                0.44

                Cobll1

                Cobl-like 1

                0.44

                Scrg1

                scrapie responsive gene 1

                0.45

                Matn3

                matrilin 3

                0.45

                Zfpm2

                zinc finger protein, multitype 2

                0.45

                Lims2

                LIM and senescent cell antigen like domains 2

                0.45

                Gpr64

                G protein-coupled receptor 64

                0.45

                Ptprz1

                protein tyrosine phosphatase, receptor type Z, polypeptide 1

                0.46

                Hhip

                Hedgehog-interacting protein

                0.46

                Eps8

                epidermal growth factor receptor pathway substrate 8

                0.46

                Heph

                hephaestin

                0.47

                Sesn1

                sestrin 1

                0.47

                Ctf1

                cardiotrophin 1

                0.47

                Zfp612

                zinc finger protein 612

                0.48

                Wdr40b

                WD repeat domain 40B

                0.48

                Dkk1

                dickkopf homolog 1 (Xenopus laevis)

                0.48

                Ogt

                O-linked N-acetylglucosamine (GlcNAc) transferase

                0.48

                Ddc

                dopa decarboxylase

                0.48

                Adam17

                a disintegrin and metallopeptidase domain 17

                0.48

                Rdhe2

                short chain dehydrogenase reductase 9

                0.48

                Vav3

                vav 3 oncogene

                0.48

                Tmem56

                transmembrane protein 56

                0.48

                Aldh1a3

                aldehyde dehydrogenase family 1, subfamily A3

                0.48

                Zfp521

                ecotropic viral integration site 3

                0.48

                Fbxo25

                F-box only protein 25

                0.49

                Kitl

                kit ligand

                0.49

                Plagl1

                pleiomorphic adenoma gene-like 1

                0.49

                Hectd2

                HECT domain containing 2

                0.49

                Bmper

                BMP-binding endothelial regulator

                0.49

                Gprasp1

                G protein-coupled receptor associated sorting protein 1

                0.50

                Gdf10

                growth differentiation factor 10

                0.50

                Sox5

                SRY-box containing gene 5

                0.50

                One of the genes showing a strong increase in the hypertrophic zone (>6-fold) was Ptgs2, encoding cyclooxygenase 2 (Cox2), a key enzyme in the synthesis of prostaglandins. Since Ptgs2 and its products (such as prostaglandin E2) are known to play important roles in chondrocyte differentiation and skeletal remodeling [2931], we selected this gene for validation experiments. Induction of Cox2 mRNA expression in the hypertrophic zone by CNP was confirmed by real-time PCR, which showed a 10-fold increase in transcript levels (Fig. 6C). p38 inhibition did reduce the basal levels of Cox2 mRNA, but surprisingly did not affect the induction by CNP. Among the genes down-regulated by CNP was the Tnfsf11 gene, encoding RANKL, a known activator of osteoclastic resorption of bone and cartilage [32, 33]. Tnfsf11 displayed a 3.8-fold reduction in expression according to microarray analyses. Because down-regulation of Tnfsf11 could provide a molecular mechanism for the observed delay in mineralization and cartilage remodeling in response to CNP, we chose to validate its expression. Real-time PCR analysis confirmed down-regulation of Tnfsf11 mRNA levels in the hypertrophic zone by CNP (Fig 6D).

                To answer the question why the hypertrophic zone is much more responsive to CNP treatment than other zones, we examined expression of key genes in the CNP signaling pathway. Analyses of our microarray data demonstrated that the genes encoding CNP (Nppc), its signaling receptor GC-B (Npr2) and the decoy receptor (Npr3) are expressed at similar levels in all three zones of micro-dissected tibiae under control conditions (Fig. 7A). However, Prkg1 (encoding cGMP-dependent kinase I) expression is 5.9 fold higher in hypertrophic chondrocytes than in the resting/proliferative cells, and seven-fold higher in the hypertrophic versus the mineralized zone (Fig. 7A). Similarly, Prkg2 expression is 4.4-fold and 2.5-fold higher in the hypertrophic zone versus the resting/proliferative and mineralized zones, respectively (Fig. 7A). This expression pattern of key mediators of CNP signaling can explain the strong responsiveness of hypertrophic chondrocytes to CNP. In addition, our microarray data on expression of the decoy receptor Npr3 in the hypertrophic zone, while variable and thus not statistically significant, suggested that CNP strongly activates the expression of Npr3. We therefore decided to analyze its expression by real-time PCR which demonstrated a statistically significant 16-fold induction of Npr3 expression in the hypertrophic zone by CNP that was not altered by p38 inhibition (Fig. 7B). CNP did not affect Npr3 expression in the other growth plate zones.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig7_HTML.jpg
                Figure 7

                Expression patterns from microarray analyses demonstrate up-regulation of cGMP-dependent kinase genes in the hypertrophic zone. Microarray analyses of the principal players in the CNP pathway in micro-dissected tibiae cultured with and without CNP (1 μM) are shown (A). Prkg1 and Prgk2, encoding cGMP-dependent kinases I and II, were strongly up-regulated in the hypertrophic zone, irrespectively of exogenous CNP. In addition, CNP strongly stimulated expression of Npr3, the natriuretic peptide clearance receptor, in the hypertrophic zone. Real-time analysis confirmed induction of Npr3 by CNP, which primarily occurs through a p38-independent manner. Data represent means ± SD of three independent trials (p < 0.05).

                Annotation of microarray data identifies CNP-regulated pathways

                To gain insight into biological processes regulated by CNP, we employed KEGG annotation [34] on genes showing at least two-fold changes in response to CNP in the hypertrophic area (Fig. 8A). Numerous pathways were affected by CNP, most of them comprised approximately proportionally by up- and down-regulated genes. However, genes related to cell adhesion were strongly enriched in up-regulated genes, suggesting that CNP promotes cell adhesion. Most notably, CNP induced expression of genes involved in cell-cell interactions such as Icam2 (intercellular adhesion molecule 2), Cdh5 (Cadherin 5), and Esam1 (endothelial cell-specific adhesion molecule). In contrast, down-regulated genes included many genes encoding extracellular matrix molecules, for example Matn1 and Matn3 (Matrilin 1 and 3), Col9a2 (procollagen type IX, alpha 2) and Col14a1 (procollagen type XIV, alpha 1) (Table 3). In addition, up-regulated genes included three members of the TGFβ superfamily, Gdf5, Inhbb and Inhba, as well as the BMP antagonist Grem1 (Fig. 8B). Besides the TGFβ family, members of the Wnt and hedgehog signaling pathways are important regulators of cartilage differentiation, and components of these pathways were regulated by CNP. Other categories in which up-regulated genes were over-represented included tight junctions and calcium signaling, whereas pantothenate and CoA biosynthesis was one example for a pathway dominated by down-regulated genes. Finally, more transcription factor-encoding genes were up-regulated than down-regulated by CNP (Fig. 8B).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-7-18/MediaObjects/12861_2006_Article_160_Fig8_HTML.jpg
                Figure 8

                Detailed analyses of microarray data identify CNP-regulated pathways. Microarray data sets from hypertrophic areas of micro-dissected tibiae cultured with and without CNP (1 μM) were analyzed using KEGG annotations (A). Genes up- and down-regulated by CNP contributed approximately proportionally to many pathways. However, up-regulated genes dominated the cell adhesion molecules, TGFbeta and calcium signaling and tight junction categories (among others). Fold change of selected genes in the BMP/GDF, Wnt and hedgehog pathways in response to CNP is shown (as ratio of CNP to control; B). A list of transcription factor genes regulated by CNP is also shown.

                We also performed KEGG analyses of CNP-regulated genes in the other zones. In the proliferative zone, genes involved in cytokine receptor interactions were most prominent with four genes, three of each were upregulated by CNP (Table 1). The categories of cell adhesion and focal adhesion molecules were represented by three genes each (data not shown). In contrast, no category was represented by more than two genes in the mineralized zone (data not shown).

                Discussion

                Gene disruption and other studies have identified the CNP pathway as one of the most important anabolic regulators of endochondral bone growth. However, the molecular and cellular mechanisms involved are not completely understood. Here we provide multiple novel insights into these mechanisms. Most importantly, we show that the p38 MAP kinase pathway is an essential mediator of CNP effects on endochondral bone growth. Second, we identify the hypertrophic zone of the growth plate as the main target of CNP signaling, likely because of the high levels of cGMP-dependent kinase I and II expression in this zone. Third, we used genome-wide microarray analyses to identify multiple target genes potentially involved in CNP effects in cartilage.

                Earlier studies have demonstrated that CNP stimulates bone growth through enhanced proliferation, mineralization and extracellular matrix synthesis [10, 12, 13]. Our data suggest that effects of CNP on longitudinal bone growth are largely due to the expansion of the hypertrophic zone, in agreement with earlier studies [23]. This could be due, in principle, to a number of effects, such as increased rate of generation of hypertrophic chondrocytes, increased size of individual hypertrophic chondrocytes, and delayed replacement of hypertrophic cartilage by bone. It should be noted that these possibilities are not exclusive, and several or all of them can contribute to the observed effects of CNP. Multiple observations support the notion that delayed removal of hypertrophic chondrocytes is one of the mechanisms involved in CNP-induced bone growth. First, terminal hypertrophic chondrocytes at the metaphysis reach a larger size, suggesting that a delay in chondrocyte replacement by bone tissue allows for a longer period of cellular growth. Furthermore, while CNP increases the size of the entire tibia significantly, this increase is not matched by a proportional increase in the area of the mineralized region. This observation suggests that CNP delays remodeling of the metaphysis and the replacement of cartilage by bone. Finally, our microarray data show that expression of RANKL, a potent activator of osteoclastic bone resorption, in the hypertrophic zone is down-regulated by CNP. RANKL is expressed in hypertrophic cartilage [3537], where it likely stimulates the removal of hypertrophic cartilage by osteoclasts and facilitates vascular invasion and ossification. Repression of RANKL expression by CNP could thus delay these remodeling events. Experiments are under way in our laboratory to examine whether osteoclast activity is indeed reduced in CNP-treated organ cultures.

                We and others have shown important roles of p38 in hypertrophic chondrocyte differentiation in vitro and in vivo [3, 27, 3841]. Thus, it is not surprising that p38 inhibition reverses CNP effects on longitudinal growth and the expansion of the hypertrophic zone. Moreover, our data show that p38 activity is required for the repression of mineralization by CNP. These data are in agreement with a recent study showing delayed primary and secondary ossification in transgenic mice overexpressing an activated form of MKK6, an upstream activator of p38, in cartilage [27]. It should be noted, however, that other phenotypes of these mice (such as reduced proliferation and delayed hypertrophy) are not recapitulated in our studies, potentially due to altered patterns and/or levels of p38 activation in the two studies (e.g. transgenic expression of activated MKK6 under the collagen II promoter versus activation of p38 through the endogenous NPR2/cGMP signaling cascade) or because CNP acts through additional pathways besides p38. Independent of these complications, our studies provide strong evidence for a novel function of p38 signaling in maintaining hypertrophic cartilage and delaying the replacement of cartilage by bone.

                However, our data also show that p38 signaling is not required for all effects of CNP on hypertrophic cartilage. While p38 inhibition results in lower basal levels of Cox2 mRNA in chondrocytes, in agreement with observations by other studies [4244], CNP still causes a strong increase in Cox2 expression in the presence of SB202190. Similarly, Npr3 induction by CNP is independent of p38 activity. Therefore, it appears likely that p38 signaling is required to achieve and/or maintain the expanded hypertrophic zone in CNP-treated bones, but not for induction of some target genes. Studies to identify additional signaling pathways connecting CNP to Cox2 gene expression are underway in our laboratory.

                Our studies also demonstrate antagonistic roles of p38 and another MAP kinase pathway, the MEK1/2-ERK1/2 pathway. Inhibition of MEK1/2 activity results in enhanced growth of endochondral bones, with no additive or synergistic effect with CNP. While our studies were in progress, other groups showed that the MEK1/2-ERK1/2 indeed reduces endochondral bone growth in vivo [26] and that CNP inhibits ERK1/2 activity [21], in agreement with our studies. Additional studies confirmed the close and reciprocal interactions between CNP-cGMP and FGF-MEK1/2-ERK signaling [22, 23]. For example, CNP was shown to repress FGF-induced growth arrest and extracellular matrix degradation by counteracting MEK1/2 activation, while FGFs 2 and 18 suppress CNP-stimulated cGMP production [22, 23]. However, none of these studies evaluated a potential role of p38 signaling in this context. Since p38 has also been implicated in FGF signal transduction in chondrocytes [45, 46], it will be interesting to investigate whether this MAP kinase is involved in the antagonistic effects of CNP and FGF in endochondral bone growth. However, both CNP and FGFs activate p38 in chondrocytes, but they have opposing effects on the growth of endochondral bones. The role of p38 in FGF effects on chondrocytes has, to our knowledge, only been studied in cell culture, not in a three-dimensional model that allows direct assessment of bone growth. Based on our data, we don't expect that p38 activation contributes to the growth-repressing activities of FGF, but this prediction needs to be experimentally verified. Nevertheless, the fact that both FGFs and CNP activate p38 despite their opposing effects on bone growth makes it unlikely that p38 contributes to crosstalk between the two signaling systems, in contrast to ERK1/2.

                Similarly, it will be important to examine whether regulation of the different MAP kinases by CNP occurs through independent, parallel pathways, or whether they regulate each other. In addition, the pathways connecting CNP to the MAP kinase modules have not been completely resolved. For example, while it has been shown that repression of ERK activity by CNP occurs at the level of the upstream kinase Raf1 and requires cGMP-dependent kinase activity [22], the exact molecular mechanism involved has not been described.

                This is, to our knowledge, one of the first studies to use micro-dissection of mammalian endochondral bones for genome-wide expression analyses by microarrays. We chose to perform these studies after 6 days of CNP stimulation, as opposed to a short term treatment. While this approach does not allow us to distinguish direct and indirect target genes of CNP, it mimics the in vivo situation where cells are exposed to auto-/paracrine CNP signaling for extended periods. Our study should therefore identify genes that are regulated by long term exposure to CNP and are thus likely to be involved in the physiological activities of CNP in the growing skeleton. Our microarray data were confirmed by real-time PCR analyses for selected genes; these data and our earlier studies [28, 47, 48] strongly suggest that the vast majority of the expression profiles detected by our microarrays correspond to the authentic gene expression patterns. Analyses of array data as well as confirmatory real-time PCRs (e.g. for type X collagen) also demonstrated that our micro-dissection protocol results in efficient separation of different zones of the cartilage and can be used for identification of novel hypertrophy-specific genes. Moreover, these data clearly show that the hypertrophic zone of the growth plate is by far the most responsive to CNP. This responsiveness does not correlate to altered levels of mRNAs for CNP itself, its signaling receptor, or the decoy receptor in control conditions. Instead, our data suggest that the expression of cGMP-dependent kinases I and II (cGKI, II) is much higher in this zone than in the other ones, providing further evidence for a crucial role of these enzymes in CNP signal transduction. Interestingly, the expression of these two genes, as well as the Nppc and Npr2 genes, in the hypertrophic zone is not altered in response to CNP. In contrast, Npr3 expression is strongly induced by CNP in the hypertrophic zone. While this induction was not identified as significant in the microarray analyses, subsequent real-time PCR confirmed the existence of this previously unknown feedback loop that likely limits CNP effects in growing cartilage.

                Our expression data suggest that both cGKI and II are involved in mediating CNP effects on cartilage development. Studies with genetically altered mice and naturally occuring rat mutants demonstrate that cGKII is the dominating protein in chondrocytes [19, 49]; however, the cartilage phenotypes of cGKII- and CNP-deficient mice are not identical, suggesting the possibility of an additional role of cGKI in CNP signaling. Double knockout mice for both cGKI and II will be required to resolve this issue.

                Thus, our data in conjunction with published studies support a model where basal CNP signaling promotes proliferation and extracellular matrix synthesis in growth plate chondrocytes. Once cells start to differentiate, they increase their expression of cGMP-dependent kinases and their responsiveness to CNP. This results in an extension of hypertrophic chondrocyte life and a delay in osteoclast and potentially vascular invasion, thus promoting maximal growth of hypertrophic chondrocytes and endochondral bone growth. At the same time, high levels of CNP signaling induce expression of Npr3 that ultimately limits CNP effects, allowing for expression of RANKL and for remodeling of the metaphysis. Experiments are under way to examine whether this model accurately describes cellular mechanisms of CNP signaling in endochondral ossification and to identify the molecular mechanisms involved.

                Detailed analyses of our microarray data provided novel insights into biological processes regulated by CNP. CNP treatment induced the expression of several genes for cell-cell interactions in the hypertrophic area (as well as the resting/proliferating zone), while at the same time repressing genes for ECM proteins. Another process regulated by CNP is signaling by TGFβ family members. Most interestingly, CNP induces expression of Gdf5 and Grem1, both of which have been implicated in skeletal development. Loss-of-function mutations of Gdf5 have been identified as cause of reduced skeletal growth in human chondrodysplasias and brachypodism mice [50]. Interestingly, GDF5 has been shown to stimulate cell adhesion in chondrocytes [51], in agreement with our data showing increased expression of both Gdf5 and cell adhesion molecules in response to CNP. Therefore, GDF5 is an excellent candidate for mediating the anabolic effects of CNP. In contrast, Grem1 encodes a BMP antagonist that is required for limb development and controls chondrogenesis [5256]. Moreover, Grem1 expression is induced by BMP/GDF signaling [5759], suggesting that its stimulation could be secondary to increased expression of Gdf5 and/or related factors (e.g. Inhbb and Inhba) in response to CNP.

                In summary, our results identify several novel components and characteristics of CNP signaling during endochondral bone growth. Collectively, these studies lead to the novel concept that CNP acts, at least in part, by delaying the terminal steps of endochondral ossification, i.e. the replacement of hypertrophic cartilage by bone. Further tests of this model in vivo and elucidation of the mechanisms involved will not only result in improved understanding of endochondral bone development, but will also be crucial for the development of potential therapeutic applications.

                Methods

                Materials

                Timed-pregnant CD1 mice were purchased from Charles River Canada. CNP, 8-(4-cpt) cGMP, and pharmacological inhibitors were obtained from Sigma and Calbiochem. Cell culture reagents were from Invitrogen and general chemicals from VWR. All real-time PCR probes and reagents were purchased from Applied Biosystems. The phospho-MKK3/6 (Cat. number 9231) and phospho-p38 (9216) antibodies were from Cell Signaling Technologies, and the β-actin antibody was from Sigma.

                Organ Culture

                Tibiae were isolated from embryonic day 15.5 (E15.5) embryos from CD1 timed-pregnant mice (Charles River Canada) using the Stemi DV4 Stereomicroscope (Zeiss). Dissection day was considered to be day 0 and tibiae were allowed to recover from dissection overnight in serum-free α-MEM media containing 0.2% Bovine Serum Albumin (BSA), 0.5 mM L-glutamine, 40 units penicillin/mL and 40 μg streptomycin/mL as described [60]. The following morning, bones in 24-well Falcon plates were measured using an eyepiece in the Stemi DV4 Stereomicroscope and treated with CNP (0.01 to 1 μM) or BSA/HCl (1 mM) vehicle, and DMSO or U0126, PD98059, PD169316, SB202190 or SB202474 (10 μM each). Media was changed every 48 hrs beginning on day 1, and bones measured on days 1, 3, 6, and 8. Results are expressed as change in length relative to day 1. Experiments were repeated at least three times, with 4–6 bones per treatment for each trial.

                For weight determination and Alizarin Red/Alcian Blue staining, 6 bones per treatment were weighed at day 6 of culture and then placed in 4% Paraformaldehyde (PFA) in DEPC-treated PBS for overnight fixation. Subsequently, tibiae were placed in staining solution for 45–60 minutes (0.05% Alizarin Red, 0.015% Alcian Blue, 5% acetic acid in 70% ethanol). Images of stained bones were taken using a Nikon SMZ1500 dissecting microscope with Photometric CoolSNAP colour digital camera (Nikon Canada) and PTI Image Master 5 program. Stained areas in images were measured using Openlab 4.0.4 software program.

                For experiments requiring perichondrium removal, tibiae were isolated from embryonic 15.5 day embryos under three different modes: very loose dissection ensuring that perichondrium was intact, very careful dissection in which perichondrium was removed mechanically, and treatment of tibiae with dispase (3 mg/mL in PBS) for 3–5 minutes with concurrent mechanical removal of perichondrium [61, 62]. Media was changed every two days beginning on day 1 and bone lengths measured on days 1 and 6, with change in length expressed relative to day 1.

                Histology and Immunohistochemistry

                After experiment completion, tibiae were rinsed with PBS and fixed in 4% PFA overnight. Bones were then stained with mercurochrome for visualization, placed in 10% formalin solution, and sent for embedding and sectioning in the Pathology lab at University of Western Ontario Hospital or the Molecular Pathology Core Facility at the Robarts Research Institute (London, Ontario, Canada). Following sectioning, bones were stained with hematoxylin and eosin using standard protocols. For immunohistochemistry, sections were incubated with primary anti-phospho-p38 antibody (1:50 dilution) over night at 4°C. Bound antibody was visualized using the UltraVision LPValue detection system (Lab Vision) with AEC chromogen substrate (Lab Vision).

                RNA isolation from organ cultures and microarray analyses

                For experiments requiring RNA isolation from organ cultures, E15.5 tibiae were harvested and treated as described above with or without CNP and SB202190. On day 6 of treatment, tibiae were separated under a dissecting microscope into the resting/proliferative, hypertrophic, and mineralized areas. Same areas from approximately 24 bones were pooled per trial, in each of three independent trials. RNA was isolated following the RNeasy ® Lipid Tissue Extraction protocol from Qiagen (Mississauga) and RNA integrity verified using the Agilent 2100 Bioanalyzer. Microarray analyses from three trials were performed at the London Regional Genomics Centre (London, Ontario, Canada) using MOE430 2.0 Affymetrix arrays consisting of 45,000 probe sets (covering the entire mouse genome). Results were analyzed using GeneSpring 7.2 software as described [28]. Microarray data were independently filtered using GeneSpring Bioscripts quality filters for noise and one-way ANOVA testing, to eliminate genes that were not expressed or showed great variability between replicates. The remaining 5199 probes sets common to both filtering methods were used for all subsequent analyses. Lists of genes undergoing at least two-fold changes were analyzed using the Babelomics suite [63] and in particular the KEGG pathways module in the FatigoPlus tool.

                Real-Time PCR

                Real-Time PCR analysis was performed as described using Applied Biosystems 7900 HT Real-Time PCR System and TaqMan® Gene Expression Assays [28, 40, 64]. All probes (Npr3, Col2a1, Col10a1, Ptgs2, Tnfsf11 and Gapdh)were purchased from Applied Biosystems. Gene expression levels were determined using the Standard Curve quantitative method with Gapdh levels as the basis of comparison.

                Statistical Analyses

                All experiments were performed in at least three independent trials. Two-Way ANOVA (parametric) test with Bonferroni post-test were performed using the Graph Pad/Prism software. One-way ANOVA with Bonferroni post-test and paired t-tests were used when appropriate.

                List of abbreviations

                BMP: 

                bone morphogenetic protein

                cGMP: 

                cyclic guanosinemonophosphate

                cGK: 

                cGMP-dependent kinase

                CNP: 

                C-type natriuretic peptide

                Cox2: 

                cyclooxygenase 2

                GDF: 

                growth differentiation factor

                ERK: 

                extracellular signal-regulated kinase

                MEK: 

                MAP/ERK kinase

                PCR: 

                polymerase chain reaction

                RANKL: 

                receptor activator of nuclear factor kappa B ligand

                TGF: 

                transforming growth factor

                Declarations

                Acknowledgements

                H.A. was supported by Ontario Graduate Studentships, S.K. and J.R.G. by graduate student awards from the Canadian Arthritis Network, and C.G.J. by Ontario Graduate Studentships in Science and Technology and a Canadian Institute for Health Research (CIHR) Doctoral Award. This work was supported by a Canada Research Chair Award and a CIHR/The Arthritis Society New Investigator Award and operating grants from the Canadian Institutes of Health Research and The Arthritis Society to F.B.

                Authors’ Affiliations

                (1)
                CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, University of Western Ontario
                (2)
                Department of Cell Biology, University of Alabama

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