Elsevier

Bone

Volume 92, November 2016, Pages 157-167
Bone

Full Length Article
C-type natriuretic peptide restores impaired skeletal growth in a murine model of glucocorticoid-induced growth retardation

https://doi.org/10.1016/j.bone.2016.08.026Get rights and content

Highlights

  • CNP restores impaired skeletal growth due to dexamethasone in a murine model.

  • CNP restores the decreased hypertrophy of growth plate chondrocytes by dexamethasone.

  • CNP could be a therapeutic agent for the glucocorticoid-induced growth retardation.

Abstract

Glucocorticoids are widely used for treating autoimmune conditions or inflammatory disorders. Long-term use of glucocorticoids causes impaired skeletal growth, a serious side effect when they are used in children. We have previously demonstrated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. In this study, we investigated the effect of CNP on impaired bone growth caused by glucocorticoids by using a transgenic mouse model with an increased circulating CNP level. Daily administration of a high dose of dexamethasone (DEX) to 4-week-old male wild-type mice for 4 weeks significantly shortened their naso-anal length, which was restored completely in DEX-treated CNP transgenic mice. Impaired growth of the long bones and vertebrae by DEX was restored to a large extent in the CNP transgenic background, with recovery in the narrowed growth plate by increased cell volume, whereas the decreased proliferation and increased apoptosis of the growth plate chondrocytes were unaffected. Trabecular bone volume was not changed by DEX treatment, but decreased significantly in a CNP transgenic background. In young male rats, the administration of high doses of DEX greatly decreased N-terminal proCNP concentrations, a marker of CNP production. In organ culture experiments using fetal wild-type murine tibias, longitudinal growth of tibial explants was inhibited by DEX but reversed by CNP. These findings now warrant further study of the therapeutic potency of CNP in glucocorticoid-induced bone growth impairment.

Introduction

Glucocorticoid (GC)-based drugs are widely used to treat various diseases because of their immunosuppressive and anti-inflammatory effects. However, the long-term use of these drugs causes many side effects on various tissues, including bone. The most notable deleterious effects on bone associated with GCs are osteoporosis and subsequent bone fractures [1]. These side effects on bone have been proposed to be caused by manifold functions of GCs, such as suppression of the proliferation and differentiation of osteoblasts, acceleration of the apoptosis of osteoblasts and osteocytes [2], and attenuation of the function of insulin-like growth factor-1 (IGF-1), which promotes bone formation [3], [4]. Moreover, it is well-known that long-term administration of GCs to children induces growth retardation. GC-induced growth impairment correlates with the dose of GCs [5]. Although it has been reported that GCs used for physiological replacement (0.075–0.125 mg/kg/day prednisone or 0.3–0.375 mg/kg/day hydrocortisone) can induce growth retardation [6], a recent study suggests that growth impairment is actualized when the GC dose exceeds 0.2 mg/kg/day prednisone equivalents [5]. Alternate-day treatment of boys with prednisone also involves growth impairment, and the impairment persists even after the treatment is discontinued, resulting in reduction of their adult height [7]. There are many studies concerning growth retardation due to GCs. Long-term, high-dose regimen of GCs induces apoptosis, impairs differentiation, prevents proliferation of growth plate chondrocytes, and inhibits bone growth [8], [9]. GCs have direct effects on chondrocytes in growth plate and in addition, GCs impair the anabolic effects of growth hormone (GH)/IGF-1 axis on growth plate chondrocytes [10], [11]. As for treatment of GC-induced growth retardation, GH therapy has been attempted but the effect was reported to be limited [12]. Ablation of Bax, the pro-apoptotic protein, was indicated to rescue GC-induced growth retardation [13], but effective therapy for GC-induced growth retardation has not been established.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family along with atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) [14], [15]. There is growing evidence that CNP is associated with endochondral bone formation and linear growth. Mice depleted of CNP or its specific receptor, natriuretic peptide receptor 2 (NPR2), develop a severe short stature phenotype owing to their impaired endochondral bone growth, indicating that CNP/NPR2 signaling is a pivotal and physiological stimulator of endochondral bone growth [16], [17]. In contrast, transgenic mice subject to targeted overexpression of CNP in cartilage or increased levels of circulating CNP exhibit prominent skeletal overgrowth phenotype [18], [19].

In humans, biallelic loss of function mutations in the NPR2 gene cause one form of short-limbed skeletal dysplasia, acromesomelic dysplasia–type Maroteaux [20], [21]. Furthermore, monoallelic loss-of-function mutations in the NPR2 gene are reported to be related to short stature [22], [23]. In contrast, monoallelic gain-of-function mutations in the NPR2 gene cause a prominent skeletal overgrowth phenotype [24], [25]. Making use of this stimulatory effect of CNP/NPR2 signaling on skeletal growth, we are now undertaking translational research on the activation of CNP/NPR2 to restore impaired skeletal growth. Previously, we studied the stimulatory effect of CNP/NPR2 activation on impaired bone growth in achondroplasia, the most common form of skeletal dysplasia, using the relevant mouse model; we showed that either targeted overexpression of CNP or increasing the levels of circulating CNP using a transgenic approach could almost completely restore the impaired skeletal growth of achondroplastic model mice [18], [26], [27]. Furthermore, we showed that exogenous administration of synthetic CNP to these mice also restored the impaired skeletal growth [27]. We expect that CNP/NPR2 activation can be used to treat impaired skeletal growth under various conditions. In this study, we investigated the effect of CNP on GC-induced impaired skeletal growth using a mouse model subjected to high-dose GC treatment and transgenic mice with increased circulating CNP levels.

Section snippets

Animals

All experimental procedures involving animals were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University (Permit number: MedKyo07598). Care of animals and all animal experiments were conducted in accordance with the institutional guidelines of Kyoto University Graduate School of Medicine.

CNP transgenic mice under the control of human serum amyloid P component (SAP) promoter (SAP-Nppc-Tg mice) were generated in the C57BL/6J background by the method previously

The effects of increased circulating CNP on impaired growth of a GC-treated mouse model

CNP transgenic mice under the control of human serum amyloid P component (SAP) promoter (SAP-Nppc-Tg mice) have approximately twice as much circulating CNP compared with wild-type mice [19]. Therefore, we used these transgenic mice as an experimental model for treatment with CNP. As we had observed that there was no significant difference in the body length or weight between wild-type and SAP-Nppc-Tg mice at the age of 4 weeks, we planned to perform experiments using 4-week-old wild-type and

Discussion

In the present study, we examined the effect of CNP on the impaired skeletal growth in a mouse model for GC-induced growth retardation. We used SAP-Nppc-Tg mice, which have increased circulating CNP levels [19]. We could successfully restore the impaired skeletal growth of a mouse model of GC-treatment with a higher level of circulating CNP. DEX impaired the growth of cranial and appendicular bones but CNP overexpression reversed the impairment of appendicular bone growth. Skull length tended

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan; the Ministry of Education, Culture, Sports, Science and Technology of Japan (#21591176 and 26461381); and the Uehara Memorial Foundation, Tokyo, Japan.

Declaration of interest

Conflicts of interest: None.

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