This is the first report to document the changes in plasma CNP peptides in children presenting with acquired disorders of thyroid function. In keeping with the known effects of TH on skeletal growth, we found at presentation highly significant differences in HV, CNP peptides, and bone turnover (BSAP), but not in IGF-I, in hyperthyroid children when compared to those with hypothyroidism. During treatment restoring euthyroidism, changes in HV and CNP peptides were greater in hyperthyroid children where T4 and CNP/NTproCNP were closely coupled. Since equally strong relationships were found for both CNP and bio-inactive NTproCNP and the ratios of NTproCNP to CNP were unaltered, it is reasonable to assume that these relationships are based on changes in CNP secretion rather than a response to changes in peptide clearance. Taken together, these novel findings suggest important interactions of TH and CNP signaling within growth plates and show that TH can be added to the previously documented factors (which include GH, testosterone, glucocorticoids, and nutrients) impacting growth and circulating CNP forms in juveniles.
Whereas other reports document the medium- (beyond 6 mo) and long-term response of linear growth to treatment in hypothyroid children, there are few detailed prospective studies of the early response. There are none reporting the growth responses of hyperthyroid children. Our observations of the responses of the two groups reveal several novel findings. First, although height SDS (and bone age) were similarly perturbed at baseline and consistent with the well-recognized effects of excess and deficiency of TH on the skeleton, change in HV in response to treatment differed in the two groups. Decrease in both HV and plasma CNP forms was more prompt and greater in subjects with hyperthyroidism than the changes observed in subjects with hypothyroidism. Proportionate change (i.e
., at 6-wk compared to basal) in TH values (3-fold rise in hypothyroidism and 4-fold fall in hyperthyroidism), while greater in the latter group, seem insufficient to account for these different responses in HV and plasma CNP forms during treatment. Why the skeletal response of the hypothyroid group was less sensitive to change in T3 is unclear, but could relate to dysregulated signaling pathways and the disorganized appearance of the hypothyroid growth plate as reported in rodents (13
). Although the condition of the growth plate in hypothyroid children is largely unknown, there are likely to be structural changes with increased deposition of glycosaminoglycans (14
) which may reduce access of CNP to chondrocytes. Furthermore, TSH is reported to impair CNP signalling in cultured rat thyroid follicular cells (15
), raising the possibility that the bioactivity of CNP is reduced in primary hypothyroidism or at least until TSH concentrations are restored to normal. In contrast, in hyperthyroidism, the structural integrity of the growth plate is retained (13
). Since some 70% of linear growth results from the increase in cell volume of hypertrophic chondrocytes, reducing drive from T3 at this late stage of endochondral growth would be expected to have a major impact on HV and, for the reasons stated below, on plasma CNP forms as well, as shown in the current study. The failure to show a significant correlation of CNP forms with HV in the hypothyroid group likely follows from the small (and temporally variable) response in HV in these children, features which do not apply in the hyperthyroid children.
While we cannot exclude contributions to circulating concentrations of CNP forms from extra-skeletal sources, prior evidence from studies in lambs (16
) and children (5
), and more recently from GH-deficient rodent pups (17
), strongly supports the view that the growth plate or a closely related tissue is a major source. Since in vitro
and ex vivo
studies show that the integrity of T3 signaling is essential to endochondral bone growth (9
) and that lack or excess of T3 strongly impacts HV in children, the current findings are consistent with the hypothesis that T3 is one of the factors driving the production of CNP by growth plate chondrocytes. Moreover, an effect of duration and severity of the disturbed T3 production on CNP secretion is evidenced by the links between plasma NTproCNP and the degree of change in skeletal maturation. Several in vitro
studies show that T3 acts principally to promote differentiation of proliferating cells to hypertrophic chondrocytes and facilitates their progression (9
). A key pathway mediating these actions of T3 in growth plate tissues includes BMP/Wnt 4 ß-catenin activation (18
) modulated by IGF-I/P13K/Akt signaling (19
). To our knowledge, the role of CNP in the trophic action of T3 within growth plate tissues has not been examined. In this context, it is intriguing to note that the principal growth-promoting locus of CNP is also the hypertrophic chondrocyte (20
) and that the molecular events underpinning this action involve some of the same mediators, e.g
., PI3K/Akt (21
) and Wnt ß-catenin (22
). Our findings that changes in CNP forms correlate highly with T4 and HV in hyper-, but not in hypothyroidism, suggest that TH do not directly increase CNP
gene expression, but act rather by increasing the supply of pre-hypertrophic chondrocytes [the putative dominant site of NPPC
gene expression in the growth plate (23
)]. However, the dynamic changes we observed during treatment strongly suggest that CNP responds to T3 drive and is downstream of T3 signaling.
Another novel observation is the differential pattern of response in BSAP shown by the two groups during treatment. Previous studies (5
) in growing lambs have shown a concordant (but slightly delayed) response of BSAP with plasma CNP forms in keeping with the close coupling of growth plate expansion and mineralization of newly formed osteoid. This pattern is evident in the response of the hypothyroid group. However, the temporal (and directional) response to treatment in hyperthyroid children was quite different, with discordant changes in plasma NTproCNP (fall) and BSAP (rise) as euthyroid status was restored over approximately 6 wk. As well as indicating that these analytes are biomarkers of separate and distinct processes within the skeleton, the findings suggest that the BSAP response (as observed in serum) is the sum of changing activity in several different tissues, including those of bone remodeling units and membranous bone, in addition to the growth plate and newly formed osteoid (9
). In hyperthyroid adults (where growth plates are atrophic), BSAP is increased at presentation in keeping with increased bone turnover (24
) and rises further in the first 6-8 wk as normal levels of T3 are restored. Since bone density also increases (25
), this rise in BSAP (a marker of bone formation) likely reflects increasing mineralization of an osteopenic skeleton. In contrast, in hypothyroid adults, markers of bone formation appear to be less affected or unchanged (26
). Hyperthyroid children will also have varying degrees of under-mineralized bone remodeling units. Seen in this light, the rise in BSAP is not unexpected and, relative to the mass of tissue involved (osteopenic trabeculae versus
growth plate-related tissue), would be expected to mask any fall in BSAP related to reduction in endochondral bone growth. While it is tempting to view the consistent fall observed in plasma NTproCNP in hyperthyroid children as largely a response to reduced activity of growth plates, change in CNP
gene expression in osteoblasts (27
) or osteoclasts cannot be excluded until more is known of the role of CNP, if any, in bone remodeling. In this context, it will be important to study changes in plasma CNP forms accompanying disorders of bone turnover in adults, including the responses of those with thyrotoxicosis.
Consistent with previous findings in childhood hypothyroidism (28
), we found no association of serum IGF-I with T3 across the whole spectrum of TH concentrations in serum and, similarly, we found no correlation of IGF-I with HV or BSAP in either group. Since circulating IGF-I is largely produced in the liver (29
) and does not necessarily correlate with events at the growth plate, the above observations are not unexpected. In contrast, compared to the liver and other organs, the skeleton appears to make significant contributions to plasma CNP forms in youth and plasma levels are positively associated with HV. Based on previous data showing an increase of NTproCNP in response to recombinant human growth hormone therapy (7
), we conclude that CNP
expression is also downstream of the IGF-I effect within growth plate chondrocytes. These unique features of CNP suggest that measurements of plasma CNP forms could provide a useful index of growth plate function in children receiving growth-promoting therapies. Thus, in the context of thyroid-based disorders of growth, a significant and sustained rise in plasma NTproCNP during treatment of hypothyroidism is likely to herald an increase in HV. Similarly, in hyperthyroid children with accelerating HV, the finding of a fall in NTproCNP could be useful in predicting an expected slowing of HV well before formal measurements of HV become available.
In conclusion, plasma NTproCNP and CNP levels in children presenting with hyperthyroidism are higher than those of hypothyroid children of similar age. Directional changes in TH, HV, and NTproCNP are concordant in response to treatment, whereas plasma IGF-I is unaffected. Links between skeletal maturation and plasma NTproCNP at presentation, and the early NTproCNP response to treatment, provide further evidence of the participation of CNP in growth plate activity and linear growth. Together with results from previous work, these findings suggest that NTproCNP may be a useful biomarker for linear growth in children with abnormal growth patterns.