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

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.

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).

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).

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).

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).

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).

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).

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).

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.