Sex hormones in the regulation of bone and cartilage metabolism: an old paradigm and a new challenge.

The effects of estrogen on tissues such as bone, endometrium and breast have been extensively studied, and the pleitropic effects of the female sex hormone are well established. Cartilage is not generally viewed as an estrogen responsive tissue. However, several epidemiological studies, and a few recent intervention studies supports that estrogen may have a role in osteoarthritis (OA), and recent animal studies further suggests that estrogen may be involved in regulation of cartilage turnover. Accordingly the issue of chondroprotrective properties of estrogen has received increased attention in recent scientific publications. In this review, we summarize current studies indicating a role for estrogen in the regulation of cartilage turnover and development of joint diseases. We report results from in vitro and animal studies where the effects of ovariectomy and treatment with estrogen and selective estrogen receptor modulators (SERM) on cartilage erosion have been evaluated. Furthermore, we report results from assessment of the effects of estrogen and SERM in postmenopausal women which shed new light on the interactions between estrogen and joint tissues. It still remains to be established whether estrogen or SERM could find a role in prophylaxis and/or treatment of OA, and much work lies ahead. Current data reviewed in this manuscript can be considered encouraging and they raise the hope that new treatment options for OA may become available based on estrogen and, in particular, compounds acting through the estrogen receptor. However, at present hormone replacement therapy and SERMs available in clinical practice, cannot be recommended as a therapy for arthritic disease.

Regulation of stem cell differentiation by histone methyltransferases and demethylases.

The generation of different cell types from stem cells containing identical genetic information and their organization into tissues and organs during development is a highly complex process that requires defined transcriptional programs. Maintenance of such programs is epigenetically regulated and the factors involved in these processes are often essential for development. The activities required for cell-fate decisions are frequently deregulated in human tumors, and the elucidation of the molecular mechanisms that regulate these processes is therefore important for understanding both developmental processes and tumorigenesis.

Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase?

AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20. RESULTS: A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p<0.01 for high-dose vs low-dose and control groups), the severity of the disease was reduced (glucose 22.2+/-3.2 vs 16.9+/-2.6 vs 15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals. CONCLUSIONS/INTERPRETATION: The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.

Age-related de-phosphorylation of proteins in dentin: a biological tool for assessment of protein age.

Mature tooth dentin has essentially no metabolic activity, and thus post-translational modifications accumulate with aging in this tissue. In the present paper, we have studied age-related covalent changes of human dentin proteins. Dentin phosphorproteins (PP) were extracted and purified using ion exchange chromatography. Collagen was purified by CNBr cleavage and acetic acid extraction. The amino acid composition of the resultant protein preparations was determined by HPLC after post-column derivatization. Likewise the extent of aspartic acid (Asp) racemization was determined in total dentin, dentin collagen and PP. Collagen only displayed small, insignificant changes in amino acid composition and racemization with age. In contrast, PP exhibited significant age-related changes in amino acid composition, cross-linking and racemization. Thus the rate of Asp racemization in PP was 500-fold that found in collagen. Moreover, the phosphoserine (Ser(P)) content in human PP decreases dramatically with age, resulting in almost complete dephosphorylation over a life span. The loss of Ser(P) was accompanied by an increased content of the bifunctional cross-link histidinoalanine consistent with a beta-elimination pathway. The relative Ser(P) content was highly correlated with dentin age (r2 = 0.96). The Ser(P) contents may thus potentially be applied in forensic investigations to deduce human age. The possible role of covalent modifications of the protein matrix in the degradation of mineralized tissue and its implications for the age-related decline of tissue functionality is discussed.

Collagen fragments in urine derived from bone resorption are highly racemized and isomerized: a biological clock of protein aging with clinical potential.

Fragments of the alpha1 C-terminal telopeptide of type I collagen containing the sequence AHDGGR(1209-1214) (CTx) can be measured in urine as an index of bone resorption. We report here that these molecules undergo racemization and isomerization of Asp(1211) in vitro and in vivo, generating a mixture of four isomers: the native peptide form (alphaL), an isomerized form containing a beta-Asp bond (betaL), a racemized form containing a D-Asp residue (alphaD) and an isomerized/racemized form (betaD). To study these reactions at this specific site in collagen, we have employed four immunoassays, each specific for one of the isoforms, and developed HPLC methods for their separation. The kinetics of these reactions were studied in vitro under physiological conditions by incubation of synthetic AHDGGR hexapeptide or mineralized bone collagen. Reactions were found to be strongly shifted towards the beta-Asp forms and slightly in favour of the D-enantiomeric forms. CTx isomers were measured in human urine and in enzymic digests of bovine bone collagen. The results indicated that the extent of racemization and isomerization were correlated with the age and turnover of collagen. The ratios between the native and age-related forms of CTx were elevated in urine from patients with Paget's disease or osteoporosis as compared with that from healthy adults. The alphaL/alphaD CTx ratio had the highest discriminatory power (T-score=23.2; P<0.0001 and T-score=1. 5; P<0.0001 for Paget's disease and osteoporosis respectively). In conclusion, these findings indicate that an assessment of CTx ratios in urine may provide an estimate of bone turnover, aiding in the diagnosis of metabolic bone diseases.

Effect of bisphosphonates on cartilage turnover assessed with a newly developed assay for collagen type II degradation products.

BACKGROUND: Animal studies of arthritis have suggested that bisphosphonates may have chondroprotective abilities. OBJECTIVE: To evaluate the effect of bisphosphonate treatment on cartilage degradation. METHODS: Type II collagen is almost exclusively localised in cartilage, where it is the major structural component of the tissue. Hence fragments derived from this protein should represent a specific index for cartilage degradation. The urinary concentration of collagen type II C-telopeptide degradation products (CTX-II) was measured by a new immunoassay (enzyme linked immunosorbent assay (ELISA)). The serum concentration of collagen type I C-telopeptide degradation products (CTX-I), a marker of bone degradation, was also measured by ELISA. PARTICIPANTS: Two groups were studied. The alendronate group included 63 healthy postmenopausal women aged 45-54 randomly allocated to receive three years' treatment with 1 mg, 5 mg, 10 mg, or 20 mg alendronate daily or placebo. In the third year the women receiving 20 mg were switched to placebo. The ibandronate group included 119 women at least 10 years after the menopause aged <75 randomly allocated to receive 12 months' treatment with 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5 mg ibandronate daily or placebo followed by 12 months without treatment. RESULTS: 20 mg of alendronate and 2.5 and 5 mg of ibandronate treatment produced significant decreases in urinary CTX-II to about 50% of baseline. The level reached after three months of treatment remained practically constant during the following 12-36 treatment months. When treatment was withdrawn CTX-II values returned towards baseline. Serum CTX-I also decreased rapidly within three months, but to a level of about 30% of baseline. CONCLUSIONS: The urinary excretion of CTX-II, a new marker of cartilage degradation, decreases significantly in response to bisphosphonate. This suggests that bisphosphonates may have chondroprotective effects in humans. By measurement of CTX-II it should be possible to monitor the effects of drugs that potentially inhibit cartilage destruction.

Racemization and isomerization of type I collagen C-telopeptides in human bone and soft tissues: assessment of tissue turnover.

Urinary excretion of the type I collagen C-telopeptide (CTx) has been shown to be a sensitive index of the rate of bone resorption. The human type I collagen sequence A(1209)HDGGR(1214) of CTx can undergo racemization of the aspartic acid residue Asp(1211) and isomerization of the bond between this residue and Gly(1212). These spontaneous non-enzymic chemical reactions takes place in vivo in bone, and the degree of racemization and isomerization of CTx molecules may be an index of the biological age and the remodelling of bone. The aim of the present study was to investigate the degree of racemization and isomerization of type I collagen in human connective soft tissues, in order to estimate the rate of collagen turnover in adult tissues and compare it with that of bone. We also performed a systematic evaluation of the pyridinium cross-link content in adult human tissues. Using antibodies raised against the different CTx forms, we found that bone and dermis are the tissues that show most racemization and isomerization. The type I collagen of arteries, lung, intestine, kidney, skeletal muscle and heart shows significantly less racemization and isomerization than that of bone, suggesting that these soft tissues have a faster turnover than bone. We also found that pyridinoline and, to a lesser degree, deoxypyridinoline are distributed throughout the different tissues investigated. Because bone type I collagen is characterized by a high degree of both racemization/isomerization and deoxypyridinoline cross-linking, the concomitant assessment of these two post-translational modifications is likely to result in a highly specific marker of bone resorption.

Characterization of urinary degradation products derived from type I collagen. Identification of a beta-isomerized Asp-Gly sequence within the C-terminal telopeptide (alpha1) region.

The heterogeneity of urinary degradation products of C-terminal telopeptides derived from the alpha1 chain of human type I collagen was investigated and characterized. The urinary fragments characterized in this study consisted of two cross-linked (X) amino acid sequences derived from the C-terminal telopeptide (alpha1) of type I collagen. Fragments containing the sequence EXAHDGGR, with a DG site being either nonisomerized (Asp-Gly) or beta-isomerized (betaAsp-Gly), were identified. Pyridinoline was detected among the pyridinium cross-links, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that of galactosyl pyridinoline. A nonfluorescent cross-link was also found. The concentration of fragments derived from the C-terminal telopeptide region of type I collagen containing the sequence Asp-Gly (alphaCTX) and/or betaAsp-Gly (betaCTX) was measured by enzyme-linked immunosorbent assays in urine and in collagenase digests of trabecular and cortical bone of young and old origin. It was shown that the urinary ratio between such fragments, alphaCTX/betaCTX, was higher in children compared with adults and that the ratio decreased with increasing age of bone. The results indicated that the C-terminal telopeptide fragments derived from type I collagen excreted into urine originated mainly from bone. In conclusion, it is demonstrated for the first time that the C-terminal telopeptide alpha1 chain of type I collagen contains an Asp-Gly site prone to undergo beta-isomerization and that the degree of beta-isomerization of this linkage apparently increases with increasing age of bone. These findings indicate that the ratio alphaCTX/betaCTX might be clinically important in diagnosing metabolic bone diseases.

Investigation of bone disease using isomerized and racemized fragments of type I collagen.

In the collagen type I C-telopeptide an aspartyl-glycine site within the sequence AHDGGR is susceptible to molecular rearrangement. In newly synthesized collagen this site is in the native form, denoted alpha L. During aging a spontaneous reaction occurs resulting in three age-modified forms: an isomerized form (beta L) a racemized form (alpha D), and an isomerized/racemized form (beta D). In this study, we measured the urinary excretion of the four forms of C-telopeptides (CTX) in healthy adults and in patients with bone diseases. Levels of all CTX forms were higher in healthy postmenopausal women (P<0.001) compared with premenopausal controls. Levels decreased within 3 days of bisphosphonate treatment indicating that all CTX forms reflect bone resorption. In hyperthyroidism, characterized by a generalized increased bone turnover, native (alpha L) and age-modified (beta L, alpha D and beta D) forms increased to a similar extent compared to controls, resulting in normal ratios between the alpha L and age-modified forms of CTX. Conversely, in Paget's disease and prostate cancer-induced bone metastases, conditions characterized by focal increased bone turnover, alpha L CTX levels were more elevated than those of age-related CTX forms, resulting in increased ratios between native and age-modified CTX. For example, the ratio alpha L/alpha D was increased 7-fold in Paget's disease (P<0.001) and 2-fold in prostate cancer-induced bone metastases (P<0.002). In conclusion, the study suggests that in conditions with a localized alteration in bone turnover the ratio between alpha L CTX and the age-modified forms is significantly elevated. This may provide a new diagnostic and monitoring tool for diseases such as metastatic bone cancer and Paget's disease.