Sport-related differences in biomarkers of bone resorption and cartilage degradation in endurance athletes.

OBJECTIVE: By measuring urinary cross-linked N-telopeptide (NTx) as a bone resorption marker and urinary C-telopeptide of type II collagen (CTx-II) as a cartilage degradation marker, we asked whether differences in skeletal stresses in college athletes undergoing high-intensity training for diverse types of aerobic sports affect their skeletal metabolism and, if so, differentially or in unison. METHODS: The study was cross-sectional at a Division 1 college campus with 60 student athletes representing crew, cross-country running and swimming. Controls were 16 non-athlete undergraduates. Urine samples were collected for NTx and CTx-II analysis by enzyme-linked immunosorbent assay, normalizing results to creatinine. Two-way analysis of variance models and pair-wise comparisons were used to test whether biomarker levels differed by sport and the significance when adjusted for body mass index (BMI). RESULTS: NTx and CTx-II showed significant differences between groups before and after adjusting for BMI. NTx was highest in the rowers, and higher in rowers and runners than in swimmers or controls. CTx-II was significantly higher in runners than in crew, swimmers or controls, when unadjusted for BMI. After adjusting for BMI, these group differences remained significant except for runners over crew. CONCLUSION: Athletes in-training in the three sports show significant differences in these markers of bone resorption and cartilage collagen degradation. The results suggest that crew undergo the highest bone remodeling and runners the highest cartilage degradation. The results also show how these markers can vary physiologically between individuals, at extremes of skeletal exercise.

Antisense oligonucleotide treatments for psoriasis.

Antisense oligonucleotides are emerging as an exciting therapeutic strategy for treating skin diseases such as psoriasis. Potential antisense targets are proteins upregulated in psoriatic skin, in particular those associated with inflammation (intercellular adhesion molecule [ICAM]-1, IL-2 and -8), proliferation (insulin-like growth factor type I receptor [IGF-IR], epidermal growth factor) and hyperangiogenesis (vascular endothelial growth factor [VEGF]). Whereas topical application and subsequent penetration of large oligonucleotides into normal skin is problematic, the impaired barrier function of psoriatic lesions permits the uptake of antisense drugs. Studies to date indicate that topically applied antisense molecules can be delivered to target cells in the epidermis and dermis of psoriatic skin. Antisense-mediated suppression of target mRNA and protein has been demonstrated in models of human skin grafted to immunosuppressed mice and in hairless mouse models of skin inflammation. In a xenograft model of human psoriasis, treatment with repeated intradermal injections of IGF-IR antisense caused a normalisation of the epidermal hyperproliferation. This class of drug, therefore, holds much potential for the successful treatment of psoriasis in the clinical setting.

Neoepitopes as biomarkers of cartilage catabolism.

Progressive degradation of articular cartilage is a central feature of arthritis and a major determinant of long term joint dysfunction. There are no treatments able to halt the progression of cartilage destruction presently available, and monitoring the benefit of potential therapies is hampered by our inability to measure the "health" of articular cartilage. Serial radiographic assessment of joint space narrowing, the current gold standard, requires measurements over a prolonged time (1-5 years) and is prone to technical difficulties. Other strategies for evaluating cartilage degradation are needed to enable both short and long term monitoring of disease progression and response to therapy. One avenue that holds promise is the use of biomarkers that accurately reflect the degradative state of the articular cartilage. Antibodies that recognise terminal amino acid sequences generated by proteolysis at specific sites in the core protein of both aggrecan and type II collagen (neoepitope antibodies) have become available in recent years. These antibodies have been invaluable for identifying the proteinases responsible for cartilage breakdown both in vitro and in vivo. The presence of neoepitope sequences generated by specific metalloenzyme cleavage of aggrecan and type II collagen correlates well with the progression of cartilage degeneration, both in vitro and in mouse models of arthritis. Preliminary results with quantitative assays of type II collagen neoepitopes suggest that they may be useful markers of joint disease in humans. Long term studies correlating neoepitope concentration with clinical and radiographic disease are now required to validate the utility of neoepitopes as surrogate markers of cartilage degeneration and joint disease.

Proteolysis of human bone collagen by cathepsin K: characterization of the cleavage sites generating by cross-linked N-telopeptide neoepitope.

An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.

Bone turnover markers and bone density across the menopausal transition.

We measured lunbar spine and femoral neck bone mineral density (BMD); urine markers of bone resorption; serum markers of bone formation; and serum gonadotrophin, estradiol and inhibin concentrations in a population-based cohort of 281 women aged 45-57 yr. Women were classified into pre-, peri-, and postmenopausal groups, depending on menstrual bleeding patterns. Compared with premenopausal women, BMD was lower only in postmenopausal women but not in women currently using hormone replacement therapy (HRT). BMD decreased with age in the perimenopausal group. Compared with premenopausal women, perimenopausal women had 20% greater urine N-telopeptide excretion (P < 0.05) and a doubling of gonadotrophin levels (P < 0.01), whereas serum estradiol and bone formation marker concentrations were no different. Postmenopausal Women had greater levels of bone turnover markers (P < 0.0001), except free deoxypyridinoline and type I procollagen propeptide. Among postmenopausal women, bone resorption markers were lower in those using HRT. Levels of nearly all bone turnover markers were positively related to serum FSH concentrations (P < 0.0001). Overall, the major independent predictors of BMD were age, urine N-telopeptide, serum bone alkaline phosphatase, and serum, FSH, whereas urine free deoxypyridinoline was positively related to BMD in pre- and perimenopausal women. In conclusion, the perimenopause is associated with elevated bone resorption rates and declining BMD, and factors in addition to estrogen deficiency may also contribute to the pathogenesis of postmenopausal osteoporosis.

1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose-response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0.5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67.4 +/- 8.8 fmol/mg protein in vehicle-treated cells to 96.7 +/- 12.4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1.08 +/- 0.10 nmol/l for 1,25-(OH)2D3-treated and 0.97 +/- 0.11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 micrograms/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.