Recombinant Human TSH Modulates In Vivo C-Telopeptides of Type-1 Collagen and Bone Alkaline Phosphatase, but Not Osteoprotegerin Production in Postmenopausal Women Monitored for Differentiated Thyroid Carcinoma†
Department of Clinical and Experimental Medicine, “F. Magrassi & A. Lanzara,” Second University of Naples, Naples, Italy
In women monitored for thyroid carcinoma, short-term stimulation with rhTSH induced an acute decrease in serum C-telopeptides of type-1 collagen and an increase in serum BALP levels without any effect on OPG production. The inhibitory effect of TSH on bone resorption occurred only in postmenopausal women who showed low BMD and a high bone turnover rate as an effect of L-thyroxine suppressive therapy.
Introduction: It has been recently shown that thyrotropin (TSH) has an inhibitory activity on skeletal remodeling in in vitro conditions. Here, we have aimed at evaluating whether TSH has similar effects in vivo. For this purpose, we have evaluated the sequential profile of serum bone metabolism markers during acute stimulation with recombinant human TSH (rhTSH) in thyroidectomized women monitored for thyroid carcinoma.
Materials and Methods: The study group included 66 thyroidectomized patients, of whom 38 were premenopausal and 28 postmenopausal, who underwent routine rhTSH-assisted whole body radioactive iodine scanning for differentiated thyroid carcinoma. The patients were sequentially evaluated for TSH, free triiodothyronine (FT3), free thyroxine (FT4), bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), and osteoprotegerin (OPG) levels during rhTSH stimulation. The samples were drawn just before and 2 and 7 days after the first administration of rhTSH. BMD was evaluated by ultrasonography at baseline. Seventy-one healthy women (41 premenopausal and 30 postmenopausal) acted as a control group.
Results and Conclusions: At study entry, all patients had subclinical thyrotoxicosis as effect of L-thyroxine (L-T4) treatment. The patients had higher serum CrossLaps and OPG levels and lower BMD than healthy subjects. Postmenopausal patients showed comparable serum FT4 and FT3 concentrations with those found in premenopausal patients. However, postmenopausal patients showed higher serum CrossLaps (p < 0.001), OPG (p = 0.03), and BALP (p < 0.001) levels and lower BMD (p < 0.001) than those measured in premenopausal patients. Two days after the first administration of rhTSH, all patients had serum TSH values >100 mUI/liter. At this time, serum CrossLaps levels decreased significantly (p < 0.001) and BALP values increased (p = 0.001) with respect to the baseline values in postmenopausal but not in premenopausal patients. rhTSH did not induce any significant change in serum OPG values either in premenopausal or in postmenopausal patients. One week after the first rhTSH administration, serum CrossLaps values decreased again to values comparable with those measured at baseline, whereas serum BALP values remained high. This study shows that subclinical thyrotoxicosis is accompanied by high bone turnover rate with an increase in serum OPG levels compared with euthyroid healthy subjects. Acute increase in serum TSH levels is accompanied by a reversible inhibition of bone resorption. This effect is characterized by a decrease in serum CrossLaps and an increase in BALP levels without any evident effect on OPG production. The activity of TSH occurs specifically in postmenopausal women in whom the negative effects of L-T4 suppressive therapy on bone mass and metabolism are more marked compared with premenopausal women.
IT IS WELL known that untreated hyperthyroidism is associated with osteoporosis and an increased fracture risk as a direct effect of active thyroid hormones on bone cells.(1–6) Thyroid hormones induce high bone turnover state in bone, leading to a decreased structure integrity of the skeleton.(6) The increase in osteotropic cytokines, as interleukin-6 and interleukin-8, could play a role in mediating the effects of thyroid hormones on bone.(7) Moreover, we have recently shown that the excess in thyroid hormones is accompanied by an increase in production of osteoprotegerin (OPG),(8) which is an important cytokine involved in the modulation of bone resorption and formation.(9–11)
It has also been emphasized that thyrotropin (TSH) may exert a critical role in skeletal remodeling by an interaction with the specific receptor expressed on bone cells.(12) In experimental animals, the reduction in expression of the TSH receptor leads to the development of osteoporosis, providing evidence for a suppressing effect of this hormone on bone turnover.(12) To our knowledge, there are no studies showing similar effects of TSH in humans. A physiological activity of TSH on bone cells seems to be unlikely because of the weak expression of the TSH receptor in human osteoblasts.(13) However, it is unclear whether TSH suppression or TSH increase, as they occur in thyrotoxicosis and hypothyroidism, may have any effect on bone metabolism.(14–19) In both pathological conditions, in fact, the abnormal serum thyroid hormone levels hinder evaluation of the specific effects of TSH on bone function.
These considerations prompted us to evaluate the sequential profile of serum bone metabolism markers during acute stimulation with recombinant human TSH (rhTSH) in women monitored for thyroid carcinoma. Such a model allowed us to evaluate the effects of TSH on bone turnover in vivo, independent of any modification in serum thyroid hormone concentrations. In fact, the patients were thyroidectomized and they remained on the same L-thyroxine (L-T4) dosage for the whole period of TSH stimulation.
MATERIALS AND METHODS
The study group included 66 thyroidectomized patients (all women [median age, 45 years], of whom 28 were postmenopausal) affected by thyroid carcinoma (follicular in 15 cases, papillary in 33 cases, follicular variant of papillary carcinoma in 18 cases) who underwent routine rhTSH- (Thyrogen; Genzyme Transgenics Corp., Cambridge, MA, USA) assisted whole body radioactive iodine scanning from January 2003 to March 2004 (Table 1). The patients were selected according to the following inclusion criteria: (1) female gender; (2) subclinical thyrotoxicosis (suppressed serum TSH and serum free triiodothyronine [FT3] and free thyroxine [FT4] in the normal range) as an effect of L-T4 treatment; (3) no chronic diseases in addition to thyroid carcinoma; and (4) no treatment with drugs influencing either bone metabolism or immune system in the last year before enrollment. At study entry, all patients had normal parathyroid function (Table 1).
The rhTSH was administered, and the whole body scan (WBS) was performed according to standard procedures.(20,21) Two doses of 0.9 mg Thyrogen, administered intramuscularly, were given once daily for the first 2 days. Twenty-four hours later, 2 mCi of123iodine was administered orally, and WBS was performed 24 h later. Patients were categorized as cured or metastatic on the basis of current clinical criteria.(22)
The patients were sequentially evaluated for TSH, FT3, FT4, bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), and OPG levels during rhTSH stimulation. For this study, the samples were drawn just before and 2 and 7 days after the first administration of rhTSH. Blood samples were collected according to the ethical guidelines between 9:00 and 11:00 a.m., after a 12-h fast, and frozen at −40°C. At baseline, all patients were also evaluated for BMD. This evaluation was performed by phalangeal ultrasonography (DBM Sonic 1200; Igea), which measured the amplitude-dependent speed of sound (ADSoS) transmission through the metaphysic of the proximal phalanx. Measurements were performed on the nondominant hand of the subject at the distal metaphysis of the first phalanges of the last four fingers. The value of the ADSoS was reported as the mean value of the four measurements. This area of the phalanx consists of a large amount of trabecular bone under a layer of corticalis. Loss of bone mass produced a decrease in ADSoS.(23,24) For this parameter, we also calculated the Z score value of the patients on the basis of the ADSoS values in the reference population,(23) according to the following formula:
Seventy-one healthy women, without thyroid and any systemic disease, were recruited from the University Hospital staff and relatives as a control group. The age of these subjects was comparable with that of patients (Table 1). Moreover, the proportion of pre- and postmenopausal women was not different between the two groups (Table 1). The study was approved by the Ethical Committee, and the enrolled subjects gave informed consent to the study.
Serum FT4 and FT3 were tested by double antibody RIA (Technogenetics, Milan, Italy), whereas serum TSH was assayed by an immunoradiometric method (DIA-Sorin). Samples were assayed in duplicate for each hormone. The detection limits of the assays and the intra- and interassay variation expressed as CVs were 1.2 pM, 2.9%, and 4.0% for FT3; 1.3 pM, 3.0%, and 5.6% for FT4; and 0.05 mU/liter, 3.1%, and 4.1% for TSH, respectively. In our laboratory, normal values were 3.8-7.7 pM for FT3, 9.0-21.5 pM for FT4, and 0.3-3.5 mU/liter for TSH. Plasma intact parathyroid hormone (PTH) was measured by a two-site immunoradiometric assay (Elsa-PTH; CIS BioInternational, Schering S.A.) with a sensitivity and intra- and interassay CVS of 2 ng/liter, 3.2%, and 5.0%, respectively. The reference range of PTH was 11–78 ng/liter. OPG was assayed by an enzyme immunoassay (Biomedica Gruppe, Wien, Austria). The detection limit of the assay was 0.14 pM, whereas the intra- and interassay CVs were 6.7% and 8.9%, respectively. CrossLaps were assayed by ELISA method (Nordic Bioscience Diagnostic A/S, Herlev, Denmark). The detection limit of the assays and the intra- and interassay assay variations expressed as CVs were 0.01 ng/ml, 5.3%, and 6.3%, respectively. In our laboratory, the reference ranges of CrossLaps values were 0.05-0.45, 0.05-0.55, and 0.1-0.80 ng/ml for men, premenopausal women, and postmenopausal women, respectively. Serum BALP was assayed by immunoradiometric method (Tandem-T Ostase; Beckman Coulter, Milan, Italy). The detection limit and intra- and interassay CVs were 0.5 μg/liter, 4.5%, and 7.0%, respectively. In our laboratory, the reference ranges of BALP values were 5.0-18.5, 3.5-14.0, and 7.0-22.0 μg/liter for men, premenopausal women, and postmenopausal women, respectively. Serum CrossLaps, BALP, and OPG levels were evaluated sequentially in healthy subjects at the same time-points as those of patients undergoing rhTSH stimulation, to assess the variability in these biochemical parameters during 1 week of follow-up. The mean variation was 10%, 15%, and 8% for CrossLaps, BALP, and OPG, respectively.
Data are expressed as median and range. Paired and unpaired data were compared using the Wilcoxon and Mann-Whitney tests, respectively. Multiple comparisons were made by the Friedman and Kruskal Wallis tests, with posthoc Bonferroni's correction. Frequencies were compared using the χ2 test, with Fisher's correction when appropriate. Correlation between variables was sought using Spearman's correlation coefficient (ρ). Statistical significance was assumed when p ≤ 0.05.
Bone metabolism markers at baseline
At the study entry, all patients had subclinical thyrotoxicosis (suppressed serum TSH with FT4 and FT3 values in the normal range) as an effect of L-T4 treatment (Table 1). The median duration of L-T4 treatment was 2.0 years (range, 1.0-4.0 years). The patients had higher serum CrossLaps, PTH, and OPG levels and lower ADSoS and ADSoS Z score compared with healthy subjects, without any significant difference in serum BALP levels (Table 1). Serum CrossLaps and BALP levels were above the reference range in 31.8% and 17.6% of patients, respectively. Serum CrossLaps, BALP, and OPG values were not significantly correlated with the duration of L-T4 treatment.
Table Table 1. Demographic and Clinical Data (Age, Serum FT4, FT3, TSH, BALP, CrossLaps, OPG, PTH, and ADSoS Transmission Through the Metaphysic of the Proximal Phalanx) of 66 Thyroidectomized Women With Thyroid Carcinoma Before Administration of Recombinant Human TSH Compared With 71 Healthy Women
We analyzed the data separately for premenopausal and postmenopausal subjects. Premenopausal patients showed higher serum CrossLaps and OPG values than those measured in the control women, without any significant difference in serum BALP and PTH levels, ADSoS, and ADSoS score (Table 2). Postmenopausal women showed higher serum CrossLaps, PTH, OPG, and BALP levels and lower ADSoS and ADSoS score than those measured in premenopausal patients and postmenopausal control subjects (Table 2). No significant difference in serum FT4 and FT3 values was found between postmenopausal and premenopausal patients (Table 2).
Table Table 2. Demographic and Clinical Data (Age, FT4, FT3, TSH, BALP, CrossLaps, OPG, PTH, and ADSoS Transmission Through the Metaphysic of the Proximal Phalanx) of 66 Thyroidectomized Women With Thyroid Carcinoma Before Administration of Recombinant Human TSH and 71 Health Women
Bone metabolism markers during rhTSH stimulation
No significant change in serum FT3 and FT4 occurred during rhTSH stimulation. Twenty-four hours after the second administration of rhTSH, all patients showed serum TSH values >100 mUI/liter. Pre- and postmenopausal patients showed different responses of bone metabolism to acute rhTSH stimulation. Serum CrossLaps levels decreased significantly (Fig. 1A), and BALP values increased (Fig. 1B) in postmenopausal patients 2 days after the first dose of rhTSH. The decrease in serum CrossLaps values was not significantly correlated with the increase in serum BALP (ρ: 0.1; p = 0.76). One week after rhTSH administration, serum TSH values were lower than those measured 2 days after rhTSH administration, and CrossLaps values returned to baseline values (Fig. 1A), whereas serum BALP concentrations remained significantly high (Fig. 1B). In premenopausal patients, serum CrossLaps and BALP values did not change significantly during rhTSH stimulation (Figs. 1A and 1B).
rhTSH did not induce any significant change in serum OPG values either in premenopausal or postmenopausal patients (Fig. 1C).
Fifty-two patients (78.8%) were classified as cured, whereas the remaining 14 patients showed biochemical and morphological evidence of metastatic/persistent disease. The response of bone markers to rhTSH was comparable in the two groups.
In the control subjects, no significant change in serum CrossLaps, BALP, and OPG values occurred during 1 week of follow-up.
This study shows that acute increase in serum TSH levels is accompanied by a reversible inhibition of bone resorption in postmenopausal thyroidectomized women undergoing L-T4 suppressive therapy for thyroid carcinoma. This effect is characterized by a decrease in serum CrossLaps and an increase in BALP levels without any evident change in serum OPG concentrations.
Recent experimental evidence proposes TSH as a suppressing hormone on bone.(12,25) TSH has direct effects on both components of skeletal remodeling mediated through a TSH receptor expressed by osteoblast and osteoclast precursors.(12) Mice lacking the TSH receptor have osteoporosis, and TSH was shown to inhibit osteoclastogenesis in in vitro conditions.(12) Here, we show in vivo that short-term stimulation with rhTSH induces an acute and reversible inhibition of bone resorption. This effect was shown only in postmenopausal women in whom the supraphysiological doses of L-T4 led to high bone turnover rate and low BMD, as assessed by ultrasonography, such as has been previously shown.(26,27)
In our patients, the effects of TSH acute administration on bone turnover consisted of a decrease in bone resorption and an increase in bone formation. Although the two processes are normally coupled, there are controversial data about their behavior in response to modifications of TSH-thyroid axis.(2,3,17,18) Guo et al.(17) reported a decrease at the same time of bone formation and resorption 2 years after normalization of serum TSH levels in hypothyroid patients previously treated by suppressive doses of L-T4. A similar finding was also found by Toivonen et al.(18) in thyroidectomized patients with thyroid carcinoma 5 weeks after L-T4 withdrawal. In the latter cases, the increase in serum TSH levels was sharper and more marked than that reported by Guo et al.(17) In contrast, Isaia et al.(2) and Pantazi and Papapetrou(3) reported a dissociation between bone formation and resorption in postmenopausal women with overt hyperthyroidism in the first weeks after correction of thyroid dysfunction, when serum thyroid hormone levels normalized and serum TSH values began to increase.
Indeed, the designs of previous studies were different from that applied by us. In the former, the increase in serum TSH levels was accompanied by a decrease in serum thyroid hormone concentrations.(3,17,18) Hence, it was not possible to discriminate clearly whether the modifications in bone turnover were caused by the increase in serum TSH or by the contemporary modifications in thyroid function. Here, we used an unique in vivo model that allowed evaluation of the extrathyroidal activity of TSH without the confounding effects induced by the change in serum thyroid hormone levels. In fact, our patients remained on the same L-T4 dosage during the rhTSH stimulation. It should be pointed out that we did not evaluate the effects of TSH in physiological conditions, because serum TSH reached quickly very high concentrations as an effect of acute pharmacological stimulation, and the patients had subclinical thyrotoxicosis as an effect of L-T4 suppressive therapy. This model provides the first evidence for an activity of TSH on bone metabolism in humans, such as has been previously shown in experimental conditions.(12) This link is strongly suggested by the fact that both bone resorption and formation significantly modified during acute rhTSH stimulation but not in the control subjects who did not undergo this protocol. The close temporal relationship between the increase in TSH and the transient decrease in CrossLaps would suggest that bone resorption was affected by TSH itself. In fact, serum CrossLaps returned to baseline concentrations when TSH decreased toward normal values. As for bone formation, we cannot exclude that TSH also had some effect on this process because it was found to be increased when TSH reached the highest values.(28) However, the fact that serum BALP levels remained high for a longer period than TSH would suggest that other factors, likely modifications in serum calcium values and PTH and vitamin D production,(3) may have contributed to this sustained effect. Although future studies will be needed to clarify this aspect, our experience suggests that, in some physiological conditions, the bone formation and resorption processes normally coupled may be dissociated, likely as an effect of an involvement of different regulatory mechanisms.
OPG and its cognate ligand, RANKL, have been identified as important factors involved in the regulation of bone metabolism mediating the paracrine signalling between osteoblast and osteoclast.(9–11) The critical importance of OPG in bone metabolism is suggested by the positive correlation between the amount of OPG gene product and bone mass in animal models.(29) In humans, OPG has been implicated in the pathogenesis of postmenopausal osteoporosis(30,31) and other metabolic diseases characterized by bone loss.(32–34) Recent evidence suggests that OPG may be also involved in the cross-talking between thyroid and bone in hyperthyroidism.(8,35) In fact, it has been shown that thyroid hormones regulate OPG production,(35) and overt hyperthyroidism is accompanied by high serum OPG levels.(8) In this study, we found an increase in circulating OPG values in patients with subclinical thyrotoxicosis. It is intriguing that serum OPG concentrations were high in both premenopausal and postmenopausal women, although only the latter had low BMD with high turnover rate. This finding would suggest that excess thyroid hormones cause an increase in OPG production even when bone turnover is just slightly increased and bone mass is still normal, as they occurred in premenopausal women taking supraphysiological doses of L-T4. If so, OPG would represents an early marker of bone impairment in the course of hyperthyroidism.
Acute rhTSH stimulation did not induce any significant change in serum OPG concentrations. This finding is not surprising because there is evidence that TSH regulates bone function by mechanisms different from those used by RANKL/OPG cytokines.(12) Moreover, we have recently shown that circulating OPG is not correlated with serum anti-TSH receptor antibodies in patients with Graves' hyperthyroidism.(8) These data provide clues that TSH does not regulate OPG production in bone, although some regulation may occur in other tissues.(36)
In conclusion, our study confirms that subclinical thyrotoxicosis induces high bone turnover rates with bone loss in postmenopausal women undergoing L-T4 suppressive therapy. Furthermore, our study shows the following. (1) Subclinical thyrotoxicosis is accompanied by an increase in serum OPG concentrations. (2) Acute increase in serum TSH levels, as produced by short-term rhTSH stimulation, is accompanied by a reversible inhibition of bone resorption. This effect is characterized by a decrease in serum CrossLaps and an increase in BALP levels without any evident effect on OPG production. (3) The inhibitory activity of TSH occurs specifically in postmenopausal women in whom the negative effects of L-T4 suppressive therapy on bone mass and metabolism are more marked compared with premenopausal women.
These preliminary results suggest that TSH could have some role in modulating bone metabolism in patients with bone loss and a high bone turnover rate. In this view, the suppressed TSH values in patients with hyperthyroidism may contribute to the bone loss induced by the excess in serum thyroid hormone levels.
This study was supported by a grant from the Second University of Naples.