The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis.

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

Periostin and transforming growth factor ß-induced protein (TGFßIp) are both expressed by osteoblasts and osteoclasts.

Periostin (Postn) and transforming growth factor ß-induced protein (TGFßIp) are two closely related extracellular matrix (ECM) proteins predominantly distributed in collagen-rich connective tissues submitted to mechanical strain, including bone and more specifically the periosteum. We have investigated the expression of Postn and TGFßIp mRNA by primary osteoblasts isolated from mouse periosteum and calvaria, or by the osteoblast-like MC3T3-E1 cell line, and by osteoclasts from mouse long bones differentiated in vitro. Secretion of Postn was measured with a specific ELISA. Postn and TGFßIp mRNA were concomitantly expressed in all three osteoblast models all along the differentiation process in a time-dependent manner. Both Postn and TGFßIp transcripts appeared early in osteoblast differentiation, and their expression increased 3-10 times in mature osteoblast cells. Expression decreased after differentiation was achieved and when the cultures mineralised. ELISA for secreted Postn showed a similar pattern. When MC3T3-E1 cells were treated with TGF-ß, Postn and TGFßIp mRNA expression and secretion were stimulated, whereas 1.25(OH)(2)D(3) had no detectable effect. Osteoclasts also expressed both Postn and TGFßIp during in vitro differentiation. Expression of both Postn and TGFßIp peaks in the early phases of osteoblast differentiation, and decreases later at the start of mineralisation. A novel finding is that Postn and TGFßIp are expressed by osteoclasts in vitro. Therefore Postn and TGFßIp proteins are potential biomarkers of early osteoblast differentiation and new bone formation.

Cathepsin K preferentially solubilizes matured bone matrix.

Bone collagen undergoes a series of enzymatic and nonenzymatic posttranslational modifications with maturation. The aim of this study was to analyze the collagenolytic efficiency of cathepsin K in relation to the extent of bone collagen age. Bone collagen posttranslational maturation was induced in vitro by preincubating bovine fetal cortical bone specimens at 37 °C for different times. The collagen enzymatic cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), the advanced glycation end product pentosidine (PEN), and the native (α) and ß-isomerized C-telopeptide (CTX) isomers were measured in each bone specimen. After extraction, bone collagen was incubated with human recombinant cathepsin K at different concentrations and its collagenolytic activity was measured by the release of hydroxyproline. To assess the affinity of cathepsin K for isomerized and nonisomerized CTX isomers, incubation with cathepsin K was also performed in the presence of various concentrations of a specific inhibitor. We showed that preincubation of bone collagen at 37 °C induces a marked increase in the bone concentration of PYD, DPD, and PEN and of CTX isomerization as reflected by the ratio of α-/ßCTX. This increase was associated with a parallel increase in the efficiency of cathepsin K to solubilize bone collagen. When cathepsin K was incubated in the presence of an inhibitor, the ß-isomerized form of collagen from 3-month- and 8-year-old bovine bone was more susceptible to degradation than the native α form. These results suggest that the collagenolytic activity of cathepsin K may be increased toward more matured bone collagen.

Serum periostin is associated with cancer mortality but not cancer risk in older home-dwelling men: A 8-year prospective analysis of the STRAMBO study.

BACKGROUND: Periostin (POSTN) regulates multiple biological behaviors of tumor cells. We studied the association of serum POSTN with mortality in home-dwelling men. METHODS: POSTN was measured at baseline using immunoassay (USCN life science, China) in 815 home-dwelling men aged 60-87 followed-up for 8 years. RESULTS: In the entire cohort, higher serum POSTN was associated with higher all-cause mortality [Hazard Ratio (HR) = 1.30 per Standard Deviation (SD), 95% Confidence Interval (CI): 1.13-1.50, p < .001] after adjustment for potential confounders. In a similar model, cancer mortality (n = 69) increased with POSTN levels (HR = 1.44 per SD, 95%CI: 1.16-1.78, p < .001). Cardiovascular mortality (n = 55) and non-cardiovascular-non-cancer mortality (n = 44) was not associated with POSTN levels (p = .28 and p = .94 respectively). In 107 men with prevalent cancer, all-cause mortality (HR = 1.93 per SD, 95%CI: 1.30-2.87, p < .005) and cancer mortality (HR = 2.07 per SD, 95%CI: 1.23-3.47, p < .01) increased with the increasing POSTN concentrations. In 613 men with incident cancer, higher POSTN concentrations were associated with higher cancer mortality (HR = 1.40 per SD, 95%CI: 1.12-1.76, p < .005) but not with the risk of cancer (HR = 1.16 per SD, 95%CI: 0.89-1.46, p = .21). CONCLUSIONS: Higher serum POSTN is associated with higher cancer mortality, but not with the cancer risk in older home-dwelling men.

Lower fracture risk in older men with higher sclerostin concentration: a prospective analysis from the MINOS study.

Sclerostin is synthesized by osteocytes and inhibits bone formation. We measured serum sclerostin levels in 710 men aged 50 years and older. Bone mineral density (BMD) was measured at the lumbar spine, hip, and distal forearm. Serum sclerostin increased with age (unadjusted r = 0.30, p < 0.001). After adjustment for age, weight, and bioavailable 17ß-estradiol, serum sclerostin correlated positively with BMD (r = 0.24 to 0.35, p < 0.001) and negatively with the levels of bone turnover markers (r = - 0.09 to - 0.23, p < 0.05 to 0.001). During a 10-year follow-up, 75 men sustained fragility fractures. Fracture risk was lower in the two upper quintiles of sclerostin combined versus three lower quintiles combined (6.1 versus 13.5%, p < 0.01). We compared fracture risk in the two highest quintiles combined versus three lower quintiles combined using the Cox model adjusted for age, weight, leisure physical activity, BMD, bone width (tubular bones), prevalent fracture, prevalent falls, ischemic heart disease, and severe abdominal aortic calcification. Men with higher sclerostin concentration had lower fracture risk (adjusted for hip BMD, hazard ratio [HR] = 0.55, 95% confidence interval [CI] 0.31 to 0.96, p < 0.05). The results were similar in 47 men with major fragility fractures (adjusted for lumbar spine BMD: HR = 0.39, 95% CI 0.17 to 0.90, p < 0.05). Men who had higher sclerostin and higher BMD (two highest quintiles) had lower risk of fracture compared with men who had lower BMD and lower sclerostin levels (three lower quintiles) (HR = 0.24, 95% CI 0.10 to 0.62, p < 0.005). Circulating sclerostin was not associated with mortality rate or the incidence of major cardiovascular events. Thus, in older men, higher serum sclerostin levels are associated with lower risk of fracture, higher BMD, and lower bone turnover rate.

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue.

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.