Hormone replacement therapy, calcium and vitamin D₃ versus calcium and vitamin D₃alone decreases markers of cartilage and bone metabolism in rheumatoid arthritis: a randomized controlled trial [ISRCTN46523456]

Rheumatoid arthritis is characterized by cartilage destruction, bone erosions, periarticular osteoporosis, and generalized bone loss resulting in increased prevalence of osteoporotic fractures [1, 2]. Some of the disease mechanisms responsible for focal bone loss may be similar to processes of generalized osteoporosis and associated with osteoclast activation [3–5].

Skeletal maintenance occurs by a tightly coupled process of bone remodeling consisting of a process of bone resorption by the osteoclasts followed by deposition of new bone by the osteoblasts. Estrogen deficiency is known to increase bone remodeling and the sustained increase in bone turnover induces a faster bone loss. Hormone replacement therapy (HRT) is known to restore this imbalance [6] and reduce the incidence of spinal and peripheral fractures in healthy women [7, 8] and also to improve bone mass in women with rheumatoid arthritis (RA) [9, 10]. Expression of estrogen receptors has been demonstrated in osteoblastic cells [11], osteoclastic cells [12], and human articular chondrocytes [13]. Estrogen decreases osteoclast formation and activity and increases apoptosis of osteoclasts [14, 15]. Furthermore estrogen also seems to have a stimulatory effect on bone formation by the osteoblasts [16]. Combined, these two effects are responsible for the bone-protective effects of estrogen and they also explain why women experience an accelerated bone loss after the menopause.

Generalized bone loss in postmenopausal women with RA will occur as a result of decreased estrogen levels accelerating bone turnover and systemic bone loss and by the inflammatory processes resulting in systemic increase of several cytokines shown to up regulate systemic bone turnover. In addition, bone loss also takes place focally as a consequence of the arthritic disease process. Markers of bone turnover provide an integrated measure of systemic turnover, and several studies have demonstrated significant elevations in, especially, resorption markers in RA [17–20]. Elevated bone resorption markers are associated with active progressive disease and decrease in bone mineral density (BMD) [18–20].

We have recently reported that treatment with HRT for 2 years in postmenopausal women with RA significantly improved BMD and also indicated a protective effect on joint destruction [10]. The aim of this randomized, controlled trial was to assess the effect of HRT in postmenopausal RA on biochemical markers of bone and cartilage turnover, the correlations between the markers and bone mass and joint damage, and the associations between changes in biochemical markers and changes in BMD and joint destruction at 2 years. The a priori assumption was that the HRT would induce a significant reduction of not only bone turnover but also cartilage turnover, indicating a structure-modifying therapeutic effect of HRT in RA.

We found that HRT reduced markers of both bone and cartilage metabolism and that the decrease in markers of bone turnover was associated with BMD gain. The type I collagen degradation marker ICTP (C-terminal telopeptide of type I collagen) and the cartilage markers CTX-II (C-terminal telopeptide fragments of type II collagen) and cartilage oligomeric matrix protein (COMP) were associated with the Larsen score at baseline. Of the markers tested, CTX-I (C-terminal telopeptide fragments of type I collagen) reflected bone turnover most sensitively.

The main objective of the present study was to analyze the effects of HRT on biochemical markers of both bone and cartilage metabolism in RA in postmenopausal women. This report is the first to show that HRT resulted in reduction of markers of both bone and cartilage turnover in women with RA. We also wanted to evaluate associations between the markers and bone mass and the Larsen score at baseline and to see if the changes in markers could predict BMD and joint destruction. There were significant correlations between several biochemical markers and bone mass and radiological status at entry into the study. Additionally, decreases in markers of bone metabolism were associated with improved bone mass. These findings support our previous results showing improved bone mass in the forearm, total hip, and lumbar spine, and also indicated a joint-protective effect of HRT in patients with progressive erosive RA [10]. The ESR also decreased and DAS 28 was decreased significantly more in the HRT group than in the controls, as has been shown in more detail in a previous work [10]. However, in view of the recent trials of HRT use among healthy postmenopausal women, showing, for example, an increased risk of cardiovascular events and breast carcinoma [29], there is a need to be cautious about HRT. It is hardly possible to generalize the results from studies of healthy postmenopausal women to patients with RA, a chronic inflammatory disease. In RA patients, the systemic inflammation seems to be more important than traditional risk factors in the development of coronary heart disease, and it may be that HRT use could find better acceptance in RA than among otherwise healthy postmenopausal women, but this issue requires further study [30].

Some limitations of the present study should be mentioned. Corrections have not been made for multiple comparisons, since the findings seem biologically reasonable and in accordance with our a priori hypothesis. Yet, one must be cautious about significances with P values at the <0.05 level, which theoretically could have occurred by chance since quite a lot of tests have been performed. It is also important to take into account that the biochemical markers that we have analyzed are not completely specific for bone or articular cartilage, because minor amounts of these markers may also be released from other tissues. However, we estimate on the basis of previous reports that they reflect bone and cartilage metabolism well enough to be able to follow and assess bone and cartilage turnover [31, 32].

Type I collagen comprises more than 90% of the organic bone matrix. Some other tissues also contain type I collagen – for example, skin, tendon, and cornea – but bone has a much higher proportion and a much higher turnover of this protein. Type I collagen has a triple-helix structure. Crosslinking by pyridinoline or deoxypyridinoline occurs between residues on the nonhelical carboxy-terminal or amino-terminal ends, termed telopeptides, and the helical portion of an adjacent collagen [33]. During osteoclastic bone resorption, cathepsin K and other proteases release peptide bound crosslinks, attached to fragments of C-terminal (CTX) or N-terminal (NTX) telopeptides [33, 34]. The crosslinks can be measured in the urine and serum as an index of bone resorption. Cathepsin K, which is a major osteoclast-derived protease, directly generates the fragments measured in the CTX-I assay. Another assay specific for fragments of the collagen type I C-telopeptide, ICTP, results primary from nonosteoclastic matrix-metalloproteinase-mediated degradation of type I collagen [34]. In accordance with the specificity of the type I collagen markers, the CTX-I assay has previously been shown to provide a significant response to antiresorptive therapies, including HRT [22, 23, 33, 35]. In addition, strong associations between levels of CTX-I and changes in this marker and subsequent change in BMD have been demonstrated [22]. The CTX-I marker has been less used in RA, but some recent studies have reported that high levels of CTX-I and CTX-II, reflecting bone and cartilage degradation, respectively, were associated with an increased risk of radiological progression in RA [18, 20, 36].

We found that CTX-I had decreased significantly in the HRT group, by 62% and 53% at 1 and 2 years, respectively. This decrease is comparable to the effect of HRT in healthy postmenopausal women [37]. A small reduction of CTX-I was also noticed in the control group at the end of first year, which possibly could be due to the treatment with calcium and vitamin D₃. CTX-I was inversely correlated with the bone mass in forearm and total hip, and both the 1- and 2-year changes in CTX-I were associated with the 2-year changes in BMD in the lumbar spine and total hip. In addition, the change in CTX-I was associated with a change in serum levels of E₂, suggesting a biological association between the two parameters. The results imply that in RA, also, serum CTX-I provides a good assessment of treatment responses to antiresorptive therapy such as HRT [18, 20, 36].

We also measured ICTP, which decreased by only 5% in the HRT group, in accord with the findings of previous studies showing similar weak responsiveness of this marker to HRT treatment in healthy postmenopausal women [23]. ICTP increased significantly in the controls, for reasons of which we are not certain. In a previous study of the effect of HRT on ICTP in RA, no change in ICTP was found [38]. These results may be considered to be in accord with the biochemical background of the markers where CTX-I is generated, whereas ICTP is destroyed by cathepsin-K-mediated degradation of the organic bone matrix [34]. At baseline, ICTP was correlated strongly with the Larsen score and to a lesser extent with the ESR, an observation that is in line with findings by others of increased serum levels of ICTP in active RA [39, 40].

Type I collagen is synthesized by osteoblasts as a precursor protein termed procollagen I. The carboxy-terminal and amino-terminal ends of procollagen I are removed during fibril formation before type I collagen is incorporated into the bone matrix. This cleavage yields two extension peptides, PICP and procollagen I amino-terminal propeptide (PINP), which are used as markers of bone formation [31, 33]. In this study, we analyzed PICP. The marker decreased significantly during the first year in the HRT group compared with controls, as has previously been found by Lems and co-workers [38]. The reduction was followed by a significant increase within the HRT group during the second year (data not shown), which might indicate an anabolic effect on the osteoblasts by HRT, in line with our previous findings of an increase in insulin-like growth factor 1 in the HRT group [41]. PICP correlated significantly with CTX-I and COMP and inversely with bone mass in the forearm at baseline. The 1- and 2-year decreases in PICP-I were associated with a reduction in COMP levels, improved BMD, and a beneficial effect on the Larsen score. The findings suggest that PICP together with CTX-I reflects the rate of bone turnover, in accord with the findings of Cortet and co-workers [40].

BSP accounts for about 10% of the noncollagenous proteins in bone and is in particular enriched at cartilage–bone interfaces. The function of the protein is not known, although a role in mineralization has been proposed [24, 42, 43]. In RA, BSP has been shown to be increased in serum, and the concentration of BSP in synovial fluid was correlated with the degree of knee joint damage in RA and was thus considered to reflect tissue breakdown [24]. In a prospective study, it was found that HRT decreased BSP in healthy postmenopausal women [43]. In our study, HRT exerted a suppressive effect, apparent as an increase of BSP in the control group but not in the HRT group. Neither the baseline levels nor the alterations in BSP were associated with bone mass or the Larsen score or its changes during the trial. This contrasts with the results for other bone markers and may be due to the restricted distribution of BSP within the tissue.

Collagen type II is the major structural protein of cartilage, comprising more than 50% of the protein in this matrix [32]. Type II collagen is synthesized by chondrocytes and degraded by proteolytic enzymes secreted by the chondrocytes and synoviocytes, including matrix metalloproteinases. The CTX-II marker derived from degradation of type II collagen was measured as an index of cartilage turnover. Previous studies have shown that CTX-II levels in the urine are elevated in patients with osteoarthritis and RA [20, 25, 36]. Lehmann and co-workers showed that antiresorptive treatment of postmenopausal women with a bisphosphonate, ibandronate, decreased not only CTX-I but also CTX-II [44]. This indicates a chondroprotective effect of this class of compounds, which has also been suggested by recent in vitro [45] and in vivo [46] studies. Of interest to our study is the fact that HRT treatment of healthy postmenopausal women has also been shown to be associated with significant lower CTX-II levels, indicating an effect of HRT on cartilage turnover [47]. In the present investigation, CTX-II in the urine decreased significantly within the HRT group, a finding that implies that HRT has a protective effect on cartilage. CTX-II was also correlated with ESR, ICTP, and the Larsen score at entry into the study, and the change in CTX-II at the end of 2 years was associated with the changes in ESR and ICTP, indicating an association with the inflammatory activity and processes of structural damage in the disease.

COMP is a 524-kDa, homopentameric, extracellular-matrix protein and it constitutes 0.5–1% of the wet weight of cartilage and is released from cartilage during the erosive process [26]. Its biological function is still unclear, but findings suggest that COMP may be involved in regulating fibril formation and maintaining the integrity of the collagen network. It was initially found in cartilage [48], but more recently it has also been found to be secreted from other tissues, such as synovial fibroblasts [49]. COMP has been shown to be increased in serum at disease onset in RA patients who developed large-joint destruction [50]. In early RA, high serum levels have recently been found to correlate also with future small-joint damage [51]. Moreover, neutralization of tumour necrosis factor α decreased serum levels of COMP in RA [52]. We show decreasing serum levels of COMP by HRT in this longitudinal study. COMP decreased significantly both within the HRT group and in comparison with the controls. As was discussed in a previous report [5], an explanation of the positive association between COMP and BMD at entry into the study could be due to a strong relation between osteoporosis and severe joint damage with decreased presence of articular cartilage and consequently reduced cartilage turnover.

Both biochemical markers reflecting cartilage metabolism were associated with the Larsen score at baseline but no correlations between changes in the markers and subsequent changes in the Larsen scores were found. One plausible reason for this lack of association may be that the Larsen did not change at all during the trial in about 40% of the patients; this fact reduces the probability of finding any significant associations between changes.

We found in this randomized, controlled trial that treatment with HRT in postmenopausal women with established RA reduced markers of bone turnover as well as of cartilage metabolism. The decrease in bone turnover markers CTX-I, ICTP, and PICP were associated with improved bone mass after 2 years, with CTX-I providing the most sensitive prognostic value. Baseline measures of ICTP and the markers of cartilage turnover, CTX-II and COMP, were correlated with the Larsen score and decreased during HRT treatment. Thus, specific biochemical markers of bone and cartilage turnover may be useful for assessing novel treatment modalities in arthritis, concerning both joint protection and prevention of osteoporosis.