Abstract
Although C-type natriuretic peptide (CNP) is crucial to post-natal endochondral growth, roles for the hormone in pubertal bone growth and the physiological effects of sex steroid substitution on CNP synthesis are not known. Using a plasma marker of CNP tissue synthesis (amino-terminal proCNP, NTproCNP), we have studied the effect of exogenous oestrogen (E2) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E2 in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E2 promptly increased plasma NTproCNP and CNP (P<0.001) to peak on day 2, and bALP (P<0.001 peaking on day 7), whereas no significant stimulation in response to T was observed in male lambs. Linear bone growth and live weight were unaffected. In adult anoestrous ewes, basal concentrations of CNP forms and bALP were lower than in ewe lambs, in keeping with skeletal maturity, but adults responded similarly to E2. Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P<0.001), were induced by E2. Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E2-induced CNP expression) were excluded by showing similar E2-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E2 stimulates plasma CNP forms and bALP in lambs and adult sheep and raise the possibility that CNP also participates in bone formation in the mature skeleton.
Introduction
C-type natriuretic peptide (CNP) belongs to a family of highly conserved peptides best known for their actions on fluid balance, blood pressure regulation and cardiac remodelling (Espiner et al. 1995, Potter et al. 2006). Distinct from atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) that are products of cardiac synthesis and secretion, CNP is synthesised in a broad range of tissues (Minamino et al. 1993, Stepan et al. 2000) and circulates in blood at levels considered insufficient to affect organ function (Hunt et al. 1994, Espiner et al. 1995). Whereas early studies highlighted antiproliferative paracrine actions of CNP within vascular tissues (Suga et al. 1992), recent findings from genetic studies clearly show that CNP has a crucial role in skeletal growth and development in both rodents (Chusho et al. 2001) and humans (Bartels et al. 2004). CNP and its receptor (NPR-B) have been shown to be expressed in human and rodent growth plates (Hagiwara et al. 1994, Chusho et al. 2001, Moncla et al. 2007). CNP in vitro strongly stimulates chondrocyte growth and expansion of growth plate tissues (Yasoda et al. 1998) and is reported to be the most potent growth factor in stimulating foetal tibial growth ex vivo (Yasoda et al. 2004).
Knowledge that the amino terminal (inactive) fragment of proCNP – amino-terminal proCNP (NTproCNP) – is likely to reflect the tissue synthesis of CNP (Prickett et al. 2005) and is readily measurable in plasma (Prickett et al. 2001) has opened up new approaches to assessing CNP's paracrine actions and role in skeletal biology. For example, the plasma concentration of NTproCNP and markers of bone formation strongly correlate with linear growth velocity in humans (Prickett et al. 2005, 2008, Olney et al. 2007), as well as during normal growth and during interventions that impact on growth velocity in lambs (Prickett et al. 2005, 2007a). Collectively these findings support the view that growth responsive tissues, such as the growth plate of long bones, are an important source of circulating NTproCNP, at least in juveniles. Factors determining NTproCNP levels in the adult are still to be clarified.
It is well known that a variety of circulating hormones have the potential to affect linear growth and bone formation (van der Eerden et al. 2003). For example, in humans sex steroids have important actions during puberty to enhance linear growth and bone accrual later in puberty and early adulthood (Eastell 2005, Wang et al. 2006). Recently, (Olney et al. 2007) we have been shown that within 4 weeks of commencing treatment, exogenous testosterone markedly stimulates plasma NTproCNP concentrations in pre-pubertal boys with delayed maturation. These findings, along with observations that plasma levels of NTproCNP are significantly raised in pubertal boys during periods of maximum growth velocity (Olney et al. 2007) support the view that increased CNP synthesis also underlies the skeletal growth changes at puberty. Whether the increase in CNP reflects a direct (acute) effect of testosterone – or follows from the subsequent increased activity of bone responsive tissues (van der Eerden et al. 2003) – is unknown. Further earlier reports (Acuff et al. 1997) indicate that CNP gene expression is rapidly enhanced by oestrogens, at least in rodent reproductive tissues. Hypothesising that sex steroid administration will acutely stimulate CNP secretion and markers of bone formation bone alkaline phosphatase (bALP) in immature juveniles, we have now studied the acute and chronic effects of exogenous E2 or T on CNP synthesis in pre-pubertal female and male lambs respectively. Finding that CNP and bALP were responsive to E2 in ewe lambs (without increase in linear growth) led us to also study the responses in mature adult ewes before and after removal of reproductive tissues.
Material and Methods
Sheep studies
Effect of oestrogens in ewe lambs
Sixteen 15-week-old pre-pubertal female Dorset Down/Coopworth cross lambs maintained on pasture were randomly allocated to receive either oestradiol impregnated or sham implants. Oestradiol treated animals (n=8) received four 30×5 mm silicon implants each containing 43.9 mg oestradiol (Compudose 400, Elanco Animal Health, New Zealand, equates to a release rate of 17 μg/kg per day). Control animals (n=8) each received four sham implants consisting of 30×4.5 mm silicone rubber tubing. Implants were placed subcutaneously in the inguinal region of the abdomen following swabbing with 70% ethanol and local anaesthetic (1 ml 2% lignocaine hydrochloride). Live weight and right metacarpal bone length (vernier calliper) were measured weekly. Blood samples were drawn prior to treatment and at 1 to 4-day intervals following implantation for 9 weeks for measurement of plasma E2, CNP, NTproCNP and bALP.
Effect of testosterone in ram lambs
Sixteen 15-week-old ram lambs were randomly allocated to receive either testosterone depot injections (50 mg testosterone cypionate IM, n=8) or vehicle control injections (cottonseed oil, n=8) at intervals of 1 week for 1 month. Live weight and right metacarpal bone length were measured weekly for a period of 9 weeks. Blood samples were drawn prior to the intervention, and then at frequent intervals (Fig. 2) for measurement of plasma T, E2, CNP, NTproCNP and bALP.
Effect of weekly injections of testosterone (closed circles, n=8) and vehicle control (open circles, n=8) in 15-week-old ram lambs. Responses in (A) plasma testosterone, (B) NTproCNP, (C) CNP, (D) bALP and (E) oestradiol concentrations are shown. Result are expressed as means±s.e.m. Significant differences from control time matched data are indicated by asterisks (*P<0.05).
Citation: Journal of Endocrinology 199, 3; 10.1677/JOE-08-0267
Effect of oestrogen in adult ewes
Sixteen anoestrous adult ewes (aged >3 years) were randomly allocated to receive either oestradiol (5.6 mg/kg live weight slow release depot, Compudose 400 implants, n=8) or sham implants (n=8) as described above. Live weight was measured at weekly intervals. Blood samples were drawn prior to the intervention and then at intervals of 1–4 days (as shown in Fig. 1E–H) for measurement of plasma E2, CNP NTproCNP and bALP.
Effect of oestradiol (closed circles, n=8) and sham (open circles, n=8) implants in (A–D) 15-week-old lambs and (E–H) adult ewes. Responses in (A and E) plasma oestradiol, (B and F) NTproCNP, (C and G) CNP and (D and H) bALP are shown. Result are expressed as means±s.e.m. Significant differences from control time matched data are indicated by asterisks (*P<0.05).
Citation: Journal of Endocrinology 199, 3; 10.1677/JOE-08-0267
Effect of oophorohysterectomy in adult ewes
In order to assess the influence of reproductive tissues on the plasma CNP response to E2, adult anoestrous ewes underwent an identical protocol as described above after sham or oophorohysterectomy. Sixteen adult ewes (aged >3 years) were randomly allocated to complete surgical removal of all uterine tissues and ovaries (HOX, n=8) or sham surgery (SS, laparotomy only, n=8). Three weeks after surgery, both groups received E2 implants. Live weight and blood sampling were performed as described above in intact ewes.
All animal studies were approved by the Lincoln University Animal Ethics Committee.
Plasma assays
Blood samples were collected in chilled standard blood collection tubes containing EDTA or lithium heparin (Vacuette; Greiner Bio-One, Kremsmuenster, Austria) and centrifuged at 4 °C; the plasma was stored at −20 °C before analysis for CNP, NTproCNP, oestradiol, testosterone (EDTA plasma) or bone ALP (heparin plasma, Ostase, Access, Beckman Coulter, Fullerton, CA, USA). Oestradiol (E2) and testosterone (T) were measured by radioimmunoassays (E2, oestradiol-2; Sorin Biomedica; T, testosterone – as described previously (Shi & Barrell 1992)). All plasma samples from individual sheep were measured in duplicate in a single assay.
RIA for NTproCNP
NTproCNP was assayed as previously described (Prickett et al. 2001, 2004) using the primary rabbit antiserum (J39) raised against NTproCNP (1–15; 100 μl 1:6000 diluted antiserum/assay tube). Peptide standards were made from synthetic human proCNP (1–19), taking into account the purity data supplied (Chiron Technologies, Victoria, Australia). Within- and between-assay coefficients of variation were 4.9 and 6.4% respectively, at 22 pmol/l. Cross-reactivity with either h NTproANP (1–30) or NTproBNP (1–21) was determined to be <0.01%.
RIA for CNP
CNP was assayed as previously described (Yandle et al. 1993) using a commercial antiserum (catalogue no. RAB-014-03; Phoenix Pharmaceuticals, Belmont, CA, USA). The rabbit antiserum raised against proCNP (82–103) shows 100% cross-reactivity with CNP-22 and human CNP-53 (Phoenix Pharmaceuticals data sheet). Within- and between-assay coefficients of variation were 3.6 and 8.3% respectively, at 7.5 pmol/l. Cross-reactivity with the natriuretic peptides hANP, hBNP32 and ovine BNP26 were <0.004, 2.3 and 1.4% respectively.
Statistical methods
Data are presented as means±s.e.m. where appropriate. Unless otherwise stated, ANOVA with repeated measures was used to assess changes in biochemical and physical measurements in sheep using time and interventions as the independent variables. When required, data were log transformed to satisfy parametric assumptions. Where significant changes were observed with ANOVA, Bonferroni post hoc analysis was used to detect differences from baseline values and control time-matched data as appropriate. Statistical significance was assumed when P<0.05.
Results
Effects of E2 in ewe lambs
The response to E2 in plasma CNP forms and bALP is shown in Fig. 1(A–D). Changes in metacarpal growth and live weight are shown in Fig. 3A and B. In keeping with pre-pubertal status, plasma E2 concentrations prior to the intervention in both control and treated lambs were low (<35 pmol/l) and remained at the level of assay detection in the control group throughout the study period. In lambs receiving oestrogen implants, plasma oestradiol peaked at 24 h (mean 1155±97 pmol/l, P<0.001) then declined progressively (Fig. 1A). Plasma E2 at 63 days (272±36 pmol/l) was still significantly increased (P<0.05) compared with levels in the control group (data not shown). Associated with these increases in E2, both plasma NTproCNP and CNP concentrations rose significantly when compared with those in control lambs (F=4.8, P<0.001 and F=5.5 P<0.001 respectively). Peak concentrations of both CNP forms occurred on day 2, some 24 h after the peak observed in plasma E2. By day 7, CNP concentrations in the E2 treated animals were not significantly different from the control lambs, whereas plasma NTproCNP concentration was still elevated relative to the control group at the completion of the study (day 63, P<0.001, data not shown).
Effect of (A and B) oestradiol (closed circles, n=8) or sham (open circles, n=8) implants in 15-week-old ewe lambs and (C and D) testosterone (closed circles, n=8) or vehicle control (open circles, n=8) injections in 15-week-old ram lambs. Responses in (A and C) metacarpal length and (B and D) live weight are shown. Result are expressed as means±s.e.m.
Citation: Journal of Endocrinology 199, 3; 10.1677/JOE-08-0267
Bone ALP (Fig. 1D) was significantly stimulated by E2, whereas no significant rise occurred in the control group. There was a significant time by treatment effect (F=3.8, P<0.001) in which bALP peaked on day 7 (Fig. 1D). Values subsequently declined to levels observed in control animals. However, at the conclusion of sampling (day 63) bALP activity was significantly lower (P<0.05) in the E2 treated group compared with the controls (data not shown).
As shown in Fig. 3, metacarpal growth velocity was similar in saline and E2 treated lambs during the first 4 weeks. Later, there was an increasing trend for metacarpal growth to fall in E2 implanted lambs. Using the pre-intervention level as the covariate, a difference in metacarpal length between the two groups on day 63 just failed to achieve significance (analysis of co-variance, P=0.066). There was no difference in live weight between E2 or sham implanted lambs during the study period (Fig. 3).
Effect of testosterone in ram lambs
The response in plasma CNP forms and bALP to weekly depot testosterone injections in 15-week-old male lambs is shown in Fig. 2(A–D). Changes in metacarpal growth and live weight are shown in Fig. 3C and D. Prior to the intervention, plasma T concentrations were in the pre-pubertal range in both control and treated lambs (median 1.5 nmol/l, range <0.3–13.3 nmol/l) and remained low in the control group for the period of study. In lambs receiving T, plasma concentrations rose promptly, as expected, after each injection achieving peaks ∼ 30–40 nmol/l (Fig. 2A). No changes were observed in plasma E2 concentrations that were <40 pmol/l in both groups (Fig. 2E).
Just prior to the intervention, plasma concentrations of NTproCNP and bALP were similar to those observed in 15-week-old ewes, whereas CNP was lower in the control group and remained so throughout the study period. In strong contrast to the effect of E2 in female lambs, there was no significant increase in either NTproCNP or CNP in T-treated lambs when compared with controls (Fig. 2B and C). Bone ALP activity was significantly lower in T-treated lambs when compared with control animals (F=2.1, P<0.05, Fig. 2D). Metacarpal growth and live weight (Fig. 3) were unaffected by T treatment.
Effects of E2 in adult ewes
As shown in Fig. 1, basal plasma concentration of E2 was low in these non-cycling ewes (<35 pmol/l) and similar to that found in ewe lambs prior to the intervention. However, in keeping with adult status and skeletal maturity, basal concentrations of NTproCNP, CNP and bALP were lower in adult ewes. Both plasma NTproCNP and CNP rose promptly in response to E2 (Fig. 1F and G) and remained significantly increased for the period of study (F=15.7 and 9.9 respectively, P<0.001 for both). Bone ALP increased threefold to peak at 15 days after E2 administration and was significantly higher (F=5.4, P<0.001) than levels in the control group. Bone ALP trended downwards in the latter half of the study period, whereas the elevated levels of plasma CNP forms were sustained.
Effect of oophorohysterectomy
As shown in Fig. 4, the response of both CNP and NTproCNP to deposit E2 was similar in sham (SS) and oophorohysterectomised (HOX) adult ewes. Increases in plasma E2 did not differ in the two groups and were similar to those observed in treated intact adult sheep. Thus, the absence of reproductive tissues (uterus and ovaries) does not affect the plasma CNP or NTproCNP response to oestrogen administration.
Effect of oestradiol implants in oophorohysterectomised adult ewes (closed circles, n=8), compared with sham operated (open circles, n=8) ewes. Responses in (A) plasma oestradiol, (B) NTproCNP and (C) CNP are shown. Result are expressed as means±s.e.m.
Citation: Journal of Endocrinology 199, 3; 10.1677/JOE-08-0267
Discussion
This is the first report showing that systemic oestrogens promptly and reproducibly stimulate circulating concentrations of both CNP and NTproCNP (and hence presumably CNP synthesis in tissues) in both immature and adult ewes. The responses occurred in the absence of change in growth velocity in lambs, and were associated with similar increases in bone specific ALP in lambs and adult sheep.
The increases we observed in plasma CNP forms were rapid (within 24 h of oestrogen administration) and consistent with an increase in CNP gene expression. We (Cameron et al. 1996) and others (Huang et al. 1996) have shown that CNP is highly expressed in brain and reproductive tissues (uterus, ovaries and placenta). To our knowledge E2 response elements in the CNP gene or promoters have not been reported. However, using adult mouse uterine tissue, Acuff et al. (1997) reported rapid (within 1 h) up-regulation of CNP in response to E2 – effects that were blocked by prior administration of actinomycin D and were dependent on nuclear oestrogen receptor activation. Concluding that E2 stimulated CNP transcription in mouse uterus, the authors (Acuff et al. 1997) postulated that similar activation of CNP by E2 may occur in other (non-reproductive) tissues. Our observation that removal of reproductive organs in the adult ewe does not affect the response of plasma CNP forms to E2 confirms this view and further indicates that enhanced CNP synthesis is maintained by high (though physiologically relevant) levels of oestrogen for prolonged periods in adult sheep.
Distinct from the response to E2, exogenous T in male lambs did not affect CNP synthesis nor stimulate bALP. The lack of response in CNP to T, evident over the 4 weeks of unchanging growth velocity, contrasts with our previous findings of increased CNP concentrations within the same period in pre-pubertal boys (Olney et al. 2007). Although caution is required when making cross species comparisons, our data suggest that T is not a direct stimulus to CNP synthesis – and that T induced CNP increase observed in pre-pubertal boys with delayed maturation reflects enhanced growth and growth plate activation rather than direct effects of the hormone on synthesis. The basis for the apparent difference in growth plate responsivity to T in the two species is unclear but could be related to the timing of the intervention and stage of pubertal maturation. Humans appear to be unique among mammals in showing a prominent and distinct surge in linear growth during adolescence following on from a period of quiescent growth in mid-childhood (Rosenfeld 2003) – which is further prolonged when maturation is delayed. By contrast, in intact ram lambs these separate phases of growth are much less obvious (Peralta et al. 1994). Possibly the increased response of growth plates to T in boys with pubertal delay is due to retention of larger numbers of resting chondrocytes – allowing their recruitment to the proliferative zones (and therefore expansion of the growth plate) under the combined effects of exogenous T and endogenous GH secretion. Of note, neither E2 (ewes) nor T (rams) increased metacarpal growth in the current study of 15-week-old lambs. These findings are in keeping with earlier studies (Peralta et al. 1994, Chanetsa et al. 2000) where increases only occurred if these agonists were administered prior to age 60–90 days. However, the lower concentration of bALP late in the treatment period in both ram and ewe lambs receiving sex steroids, and a trend for reduced growth velocity in ewe lambs, are consistent with actions of these hormones enhancing bone maturation over the course of this study. E2 is known to advance epiphysial fusion. In rabbits, raising circulating concentrations of E2 to ∼500 pmol/l (broadly similar to levels in the current study) reduces growth plate activity of the distal tibia within 2 weeks (Weise et al. 2001). In light of this, any CNP produced within the growth plate by E2 in our study would have limited time to act before chondrocyte function was reduced.
Our study was not designed to explore the source of the increased CNP secretion we observed during oestrogen treatment. Clearly a broad range of tissues outside the reproductive system express CNP (Minamino et al. 1993, Stepan et al. 2000) and could contribute to circulating levels (Charles et al. 2006). However, the close coupling of CNP and bALP responses suggests that the osteoblast may be an important source. Hitherto, most studies of CNP's skeletal actions have focused on chondrocytes within growth plates where cellular proliferation, differentiation and hypertrophy (Chusho et al. 2001, Yasoda et al. 2004, Agoston et al. 2007) are reproducible findings. Our previous in vivo observations in juveniles, linking growth velocity, plasma NTproCNP and ALP in lambs (Prickett et al. 2005) and children (Prickett et al. 2005, 2008, Olney et al. 2007) are also consistent with these findings. Over and above actions in chondrocytes, in vitro studies in rodents show that CNP transcripts are present in cells of osteoblast lineage (Suda et al. 1996, 1999) and that CNP production from these cell lines can be augmented by TGFβ (Suda et al. 1996). Further the addition of CNP to murine pre-osteoblasts, acting via NPR-B and cGMP, inhibits cell proliferation while enhancing differentiation and markers of osteoblast maturation (ALP and osteocalcin; Hagiwara et al. 1996). CNP also increases the formation of mineralised nodules. Of note, in this model the increase in ALP was delayed for 4 days and required sustained concentrations of CNP (or cGMP) in order to promote enhanced ALP mRNA expression. Our observations of similar patterns of response in bALP to sustained increases in CNP synthesis in vivo are consistent with these in vitro findings. Clearly more focused study of E2 actions on CNP synthesis within the osteoblast lineage is required, and is currently under study.
It is instructive to compare the responses of CNP forms and bALP to E2 in female lambs and adult ewes (Fig. 1). Despite a similar stimulus (plasma E2 concentration) there appears to be a biphasic response in CNP in lambs, the initial phase declining after 1 week beyond that NTproCNP alone remained elevated. This response contrasts with the similarly prompt but sustained elevation of both NTproCNP and CNP concentrations in the adult. Relevant here are the recent findings on osteocrin (Moffatt et al. 2007) – a novel endogenous and clearance receptor-specific ligand, synthesised by newly formed osteoblasts (Thomas et al. 2003) and at sites of bone remodelling in the adult skeleton (Bord et al. 2005). In binding to the natriuretic clearance receptor (NPR-C), osteocrin has the potential to increase the local concentration of CNP (Moffatt et al. 2007) but is unlikely to affect the concentration of NTproCNP (Prickett et al. 2005, Moffatt et al. 2007). Interestingly, osteocrin expression by newly formed osteoblasts is progressively inhibited over a 6-day-period by E2 (100 pmol/l), whereas its synthesis appears to be enhanced in bone tissues taken from postmenopausal women receiving depot preparations of oestrogens (Bord et al. 2005). On these grounds, differential responses of osteocrin to E2, based on the maturity of osteoblasts (Bord et al. 2005), could underlie the abbreviated (7-day) CNP response to E2 we observe in lambs. Bone ALP rose more promptly in the lambs and occurred on a background of decreasing levels (also observed in males, see Figs 1 and 2) as the skeleton matured. Further the bALP response was briefer than in adults. Possibly these differences also reflect osteoblastogenesis in metaphyseal bone (throughout the skeleton) in adults versus the completion of endochondral ossification related to growth plates in lambs. A slower and more prolonged and sustained response in CNP and ALP in the mature skeleton is in keeping with the dynamics of osteoblast recruitment from bone marrow precursors in previous studies (Samuels et al. 1999, Plant et al. 2002).
Clearly the role of CNP in organ function continues to expand as new methods allow detection of changes in tissue synthesis. Once considered primarily a regulator of vascular smooth muscle proliferation (Suga et al. 1992), more recent work shows that the hormone is intimately involved in skeletal growth, is nutrition dependent (Prickett et al. 2007a), and participates in maintaining foetal-maternal welfare (Prickett et al. 2007b). CNP also appears to have a cardioprotective role by inhibiting ventricular remodelling after cardiac injury (Pagel-Langenickel et al. 2007). Knowledge that CNP synthesis (outside the reproductive system) is E2-sensitive opens up prospects of important new roles within the skeleton and other tissues which now need to be strongly pursued.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from the New Zealand Lottery Grants Board, the Canterbury Medical Research Foundation and the Health Research Council of New Zealand.
Acknowledgements
We gratefully acknowledge expert technical assistance of Jo Anne de Ruiter and Rachael McCloy.
References
Acuff CG, Huang H & Steinhelper ME 1997 Estradiol induces C-type natriuretic peptide gene expression in mouse uterus. American Journal of Physiology 273 H2672–H2677.
Agoston H, Khan S, James CG, Gillespie JR, Serra R, Stanton LA & Beier F 2007 C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways. BMC Developmental Biology 7 18.
Bartels CF, Bukulmez H, Padayatti P, Rhee DK, van Ravenswaaij-Arts C, Pauli RM, Mundlos S, Chitayat D, Shih LY & Al-Gazali LI et al. 2004 Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maroteaux. American Journal of Human Genetics 75 27–34.
Bord S, Ireland DC, Moffatt P, Thomas GP & Compston JE 2005 Characterization of osteocrin expression in human bone. Journal of Histochemistry and Cytochemistry 53 1181–1187.
Cameron VA, Aitken GD, Ellmers LJ, Kennedy MA & Espiner EA 1996 The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta. Endocrinology 137 817–824.
Chanetsa F, Hillman LS, Thomas MG & Keisler DH 2000 Estrogen agonist (zeranol) treatment in a castrated male lamb model: effects on growth and bone mineral accretion. Journal of Bone and Mineral Research 15 1361–1367.
Charles CJ, Prickett TC, Espiner EA, Rademaker MT, Richards AM & Yandle TG 2006 Regional sampling and the effects of experimental heart failure in sheep: differential responses in A, B and C-type natriuretic peptides. Peptides 27 62–68.
Chusho H, Tamura N, Ogawa Y, Yasoda A, Suda M, Miyazawa T, Nakamura K, Nakao K, Kurihara T & Komatsu Y et al. 2001 Dwarfism and early death in mice lacking C-type natriuretic peptide. PNAS 98 4016–4021.
Eastell R 2005 Role of oestrogen in the regulation of bone turnover at the menarche. Journal of Endocrinology 185 223–234.
van der Eerden BC, Karperien M & Wit JM 2003 Systemic and local regulation of the growth plate. Endocrine Reviews 24 782–801.
Espiner EA, Richards AM, Yandle TG & Nicholls MG 1995 Natriuretic hormones. Endocrinology and Metabolism Clinics of North America 24 481–509.
Hagiwara H, Sakaguchi H, Itakura M, Yoshimoto T, Furuya M, Tanaka S & Hirose S 1994 Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B. Journal of Biological Chemistry 269 10729–10733.
Hagiwara H, Inoue A, Yamaguchi A, Yokose S, Furuya M, Tanaka S & Hirose S 1996 cGMP produced in response to ANP and CNP regulates proliferation and differentiation of osteoblastic cells. American Journal of Physiology. Cell Physiology 270 C1311–C1318.
Huang H, Acuff CG & Steinhelper ME 1996 Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide. American Journal of Physiology. Heart and Circulatory Physiology 271 H1565–H1575.
Hunt PJ, Richards AM, Espiner EA, Nicholls MG & Yandle TG 1994 Bioactivity and metabolism of C-type natriuretic peptide in normal man. Journal of Clinical Endocrinology and Metabolism 78 1428–1435.
Minamino N, Aburaya M, Kojima M, Miyamoto K, Kangawa K & Matsuo H 1993 Distribution of C-type natriuretic peptide and its messenger RNA in rat central nervous system and peripheral tissue. Biochemical and Biophysical Research Communications 197 326–335.
Moffatt P, Thomas G, Sellin K, Bessette MC, Lafreniere F, Akhouayri O, St-Arnaud R & Lanctot C 2007 Osteocrin is a specific ligand of the natriuretic peptide clearance receptor that modulates bone growth. Journal of Biological Chemistry 282 36454–36462.
Moncla A, Missirian C, Cacciagli P, Balzamo E, Legeai-Mallet L, Jouve JL, Chabrol B, Le Merrer M, Plessis G & Villard L et al. 2007 A cluster of translocation breakpoints in 2q37 is associated with overexpression of NPPC in patients with a similar overgrowth phenotype. Human Mutation 28 1183–1188.
Olney RC, Prickett TC, Yandle TG, Espiner EA, Han JC & Mauras N 2007 Amino-terminal propeptide of C-type natriuretic peptide and linear growth in children: effects of puberty, testosterone and growth hormone. Journal of Clinical Endocrinology and Metabolism 92 4294–4298.
Pagel-Langenickel I, Buttgereit J, Bader M & Langenickel TH 2007 Natriuretic peptide receptor B signaling in the cardiovascular system: protection from cardiac hypertrophy. Journal of Molecular Medicine 85 797–810.
Peralta JM, Arnold AM, Currie WB & Thonney ML 1994 Effects of testosterone on skeletal growth in lambs as assessed by labeling index of chondrocytes in the metacarpal bone growth plate. Journal of Animal Science 72 2629–2634.
Plant A, Samuels A, Perry MJ, Colley S, Gibson R & Tobias JH 2002 Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells. Journal of Cellular Biochemistry 84 285–294.
Potter LR, Abbey-Hosch S & Dickey DM 2006 Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocrine Reviews 27 47–72.
Prickett TCR, Yandle TG, Nicholls MG, Espiner EA & Richards AM 2001 Identification of amino-terminal pro-C-type natriuretic peptide in human plasma. Biochemical and Biophysical Research Communications 286 513–517.
Prickett TCR, Kaaja RJ, Nicholls MG, Espiner EA, Richards AM & Yandle TG 2004 N-terminal pro-C-type natriuretic peptide, but not C-type natriuretic peptide, is greatly elevated in the fetal circulation. Clinical Science 106 535–540.
Prickett TCR, Lynn AM, Barrell GK, Darlow BA, Cameron VA, Espiner EA, Richards AM & Yandle TG 2005 Amino-terminal proCNP: a putative marker of cartilage activity in postnatal growth. Pediatric Research 58 334–340.
Prickett TC, Barrell GK, Wellby M, Yandle TG, Richards AM & Espiner EA 2007a Response of plasma CNP forms to acute anabolic and catabolic interventions in growing lambs. American Journal of Physiology. Endocrinology and Metabolism 292 E1395–E1400.
Prickett TC, Rumball CW, Buckley AJ, Bloomfield FH, Yandle TG, Harding JE & Espiner EA 2007b C-type natriuretic peptide forms in the ovine fetal and maternal circulations: evidence for independent regulation and reciprocal response to undernutrition. Endocrinology 148 4015–4022.
Prickett TCR, Dixon B, Frampton C, Yandle TG, Richards AM, Espiner EA & Darlow BA 2008 Plasma aminoterminal pro C-type natriuretic peptide in the neonate: relation to gestational age and post natal linear growth. Journal of Clinical Endocrinology and Metabolism 93 225–232.
Rosenfeld RG 2003 Insulin-like growth factors and the basis of growth. New England Journal of Medicine 349 2184–2186.
Samuels A, Perry MJ & Tobias JH 1999 High-dose estrogen induces de novo medullary bone formation in female mice. Journal of Bone and Mineral Research 14 178–186.
Shi ZD & Barrell GK 1992 Requirement of thyroid function for the expression of seasonal reproductive and related changes in red deer (Cervus elaphus) stags. Journal of Reproduction and Fertility 94 251–259.
Stepan H, Leitner E, Bader M & Walther T 2000 Organ-specific mRNA distribution of C-type natriuretic peptide in neonatal and adult mice. Regulatory Peptides 95 81–85.
Suda M, Tanaka K, Fukushima M, Natsui K, Yasoda A, Komatsu Y, Ogawa Y, Itoh H & Nakao K 1996 C-type natriuretic peptide as an autocrine/paracrine regulator of osteoblast. Evidence for possible presence of bone natriuretic peptide system. Biochemical and Biophysical Research Communications 223 1–6.
Suda M, Komatsu Y, Tanaka K, Yasoda A, Sakuma Y, Tamura N, Ogawa Y & Nakao K 1999 C-type natriuretic peptide/guanylate cyclase B system in rat osteogenic ROB-C26 cells and its down-regulation by dexamethazone. Calcified Tissue International 65 472–478.
Suga S, Nakao K, Kishimoto I, Hosoda K, Mukoyama M, Arai H, Shirakami G, Ogawa Y, Komatsu Y & Nakagawa O 1992 Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells. Circulation Research 71 34–39.
Thomas G, Moffatt P, Salois P, Gaumond MH, Gingras R, Godin E, Miao D, Goltzman D & Lanctot C 2003 Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype. Journal of Biological Chemistry 278 50563–50571.
Wang Q, Alen M, Nicholson PH, Halleen JM, Alatalo SL, Ohlsson C, Suominen H & Cheng S 2006 Differential effects of sex hormones on peri- and endocortical bone surfaces in pubertal girls. Journal of Clinical Endocrinology and Metabolism 91 277–282.
Weise M, De-Levi S, Barnes KM, Gafni RI, Abad V & Baron J 2001 Effects of estrogen on growth plate senescence and epiphyseal fusion. PNAS 98 6871–6876.
Yandle TG, Fisher S, Charles C, Espiner EA & Richards AM 1993 The ovine hypothalamus and pituitary have markedly different distributions of C-type natriuretic peptide forms. Peptides 14 713–716.
Yasoda A, Ogawa Y, Suda M, Tamura N, Mori K, Sakuma Y, Chusho H, Shiota K, Tanaka K & Nakao K 1998 Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway. Journal of Biological Chemistry 273 11695–11700.
Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, Kurihara T, Rogi T, Tanaka S & Suda M et al. 2004 Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nature Medicine 10 80–86.
