Sclerostin: current knowledge and future perspectives.

In recent years study of rare human bone disorders has led to the identification of important signaling pathways that regulate bone formation. Such diseases include the bone sclerosing dysplasias sclerosteosis and van Buchem disease, which are due to deficiency of sclerostin, a protein secreted by osteocytes that inhibits bone formation by osteoblasts. The restricted expression pattern of sclerostin in the skeleton and the exclusive bone phenotype of good quality of patients with sclerosteosis and van Buchem disease provide the basis for the design of therapeutics that stimulate bone formation. We review here current knowledge of the regulation of the expression and formation of sclerostin, its mechanism of action, and its potential as a bone-building treatment for patients with osteoporosis.

Sclerostin in mineralized matrices and van Buchem disease.

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Bone morphogenetic proteins and their antagonists: the sclerostin paradigm.

Sclerosteosis and Van Buchem disease are two closely related sclerosing disorders, characterized by progressive bone thickening due to increased bone formation. Sclerosteosis is due to premature termination mutations in the SOST gene, whereas Van Buchem disease has been associated with a 52 kb deletion downstream of the SOST gene that most likely inhibits SOST gene transcription. The gene product, sclerostin, is an osteocyte-expressed negative regulator of bone formation with amino acid sequence similarity with the DAN family of secreted glycoproteins, that share the capacity to antagonize bone morphogenetic protein (BMP) activity. The exact mechanism, however, by which sclerostin inhibits osteoblastic bone formation is still uncertain. While it binds BMPs and antagonizes their bone forming capacity, it cannot antagonize all BMP responses. Sclerostin's mechanism of action is, therefore, distinct from that described for classical BMP antagonists. The restricted expression of sclerostin and the exclusive bone phenotype of good quality of patients with sclerosteosis or Van Buchem disease provide the basis for the development of therapeutics that stimulate bone formation, such as, for example, an antibody against sclerostin.

Use of bone morphogenetic proteins in traumatology.

An estimated 5-10% of all fractures show impaired healing, leading to delayed union, or non-union. Chemical, or physical methods to accelerate bone healing are of great interest to the orthopaedic and trauma community. Research over the last 20 years has established that successful fracture healing is steered by specific growth factors. Of these, the bone morphogenetic proteins (BMPs) are probably the most important. The signalling pathway of these proteins is tightly regulated, overseeing a finely orchestrated cascade of events that occur after a fracture. The promising results of BMPs in preclinical studies have recently cleared the way for their use in specific fractures, or non-unions in clinical practice. The purpose of this work is to give a brief overview of BMPs and to review the clinical data currently available on the use of BMPs in fracture healing.

Interleukin 17 synergises with tumour necrosis factor alpha to induce cartilage destruction in vitro.

BACKGROUND: Interleukin 17 (IL17) is produced by activated T cells and has been implicated in the development of bone lesions and cartilage degradation in rheumatoid arthritis (RA). OBJECTIVE: To determine whether IL17, alone or together with tumour necrosis factor alpha (TNFalpha), induces cartilage destruction in vitro. METHODS: Fetal mouse metatarsals stripped of endogenous osteoclast precursors were used to study the effect of IL17 on cartilage degradation independently of osteoclastic resorption. Cartilage destruction was analysed histologically by Alcian blue staining. RESULTS: IL17 alone, up to 100 ng/ml, had no effect on the cartilage of fetal mouse metatarsals. IL17 (>/=0.1 ng/ml), however, induced severe cartilage degradation when given together with TNFalpha (>/=1 ng/ml). The cytokine combination decreased Alcian blue staining, a marker of proteoglycans, throughout the metatarsals and induced loss of the proliferating and early hypertrophic chondrocyte zones. TNFalpha alone also decreased Alcian blue staining, but not as dramatically as the cytokine combination. In addition, it did not induce loss of chondrocyte zones. Treatment with inhibitors of matrix metalloproteinase (MMP) activity and nitric oxide synthesis showed that MMP activity played a part in cartilage degradation, whereas nitric oxide production did not. CONCLUSIONS: IL17, together with TNFalpha, induced cartilage degradation in fetal mouse metatarsals in vitro. IL17 may, therefore, participate in the development of cartilage destruction associated with RA by enhancing the effects of TNFalpha and may provide a potential therapeutic target.

Exposure of KS483 cells to estrogen enhances osteogenesis and inhibits adipogenesis.

Osteoblasts and adipocytes arise from a common progenitor cell in bone marrow. Whether estrogen directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. Using a mouse clonal cell line KS483 cultured in charcoal-stripped fetal bovine serum (FBS), we showed that 17beta-estradiol (E2) stimulates the differentiation of progenitor cells into osteoblasts and concurrently inhibits adipocyte formation in an estrogen receptor (ER)-dependent way. E2 increased alkaline phosphate (ALP) activity and nodule formation and stimulated messenger RNA (mRNA) expression of core-binding factor alpha-1 (Cbfa1), parathyroid hormone/parathyroid hormone-related protein receptors (PTH/PTHrP-Rs), and osteocalcin. In contrast, E2 decreased adipocyte numbers and down-regulated mRNA expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)2, adipocyte protein 2 (aP2), and lipoprotein lipase (LPL). Furthermore, the reciprocal control of osteoblast and adipocyte differentiation by E2 was observed also in the presence of the adipogenic mixture of isobutylmethylxanthine, dexamethasone, and insulin. Immunohistochemical staining showed that ERalpha and ERbeta were present in osteoblasts and adipocytes. A new mouse splice variant ERbeta2 was identified, which differed in two amino acid residues from the rat isoform. E2 down-regulated mRNA expression of ERalpha, ERbeta1, and ERbeta2. The effects of E2 are not restricted to the KS483 cell line because similar results were obtained in mouse bone marrow cell cultures. Our results indicate that estrogen, in addition to stimulation of osteogenesis, inhibits adipogenesis, which might explain the clinical observations that estrogen-deficiency leads to an increase in adipocytes.

Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation.

In this study, we examine the role of bone morphogenetic protein (BMP) signaling during differentiation of the murine preosteoblastic KS483 cell line, which formed alkaline phosphatase (ALP)-positive and mineralized nodules during a 3 week culture period. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of various BMPs (BMP-2, -3, -4, -6, -7, and -8A and -8B), BMP type I and II receptors (ALK2, ALK3, ALK4, BMPR-II, and ActR-IIA and -IIB), BMP antagonists (DAN, gremlin, chordin, cerberus, noggin, and tsg), and Smads 1-8. mRNA expression of these genes did not change during differentiation, except for BMP-3, BMP-8a, and noggin. BMP-3 increased gradually, particularly in the matrix formation phase; BMP-8a was induced from the onset of matrix maturation and mineralization, in parallel to the expression of osteocalcin; and noggin tended to decline during the mineralization phase. Treatment of KS483 cells with the BMP antagonists noggin or soluble truncated BMPR-IA, either continuously or during distinct periods of osteoblast differentiation; that is, matrix formation or matrix maturation and mineralization phase, decreased ALP-positive and mineralized nodule area independent of the phase of osteoblast differentiation. Notably, the antagonists inhibited mineralization of already existing nodules. Similarly, BMP-4 stimulated differentiation not only at the beginning of the culture period, but also at late stages of differentiation. These data indicate that autocrine BMP signaling is involved in KS483 osteoblastic differentiation not only during the early phase of differentiation, but also during matrix maturation and mineralization. The different expression patterns of components of BMP signaling in the KS483 cells suggest distinct functions of individual BMPs during osteoblast differentiation. In summary, our data suggest that BMP activity is required not only for initiation of osteoblast differentiation and further development of early osteoblasts, but is also involved in late-stage osteoblast differentiation and matrix mineralization.

Effect of interleukin-17 on nitric oxide production and osteoclastic bone resorption: is there dependency on nuclear factor-kappaB and receptor activator of nuclear factor kappaB (RANK)/RANK ligand signaling?

Interleukin-17 (IL-17) is a proinflammatory cytokine produced exclusively by activated memory T cells and has recently been found to stimulate osteoclastic resorption. Like other proinflammatory cytokines, IL-17 may affect osteoclastic bone resorption indirectly via osteoblasts, possibly by mechanisms previously reported for chondrocytes that respond in very similarly to osteoblasts. As in chondrocytes, but only in combination with tumor necrosis factor-alpha (TNF-alpha), IL-17 induced nitric oxide (NO) production in osteoblastic cells and fetal mouse metatarsals by a nuclear factor-kappaB (NF-kappaB)-dependent mechanism. This effect was associated with elevated mRNA levels of the NF-kappaB isoforms RelA and p50. In fetal mouse metatarsals, IL-17 stimulated osteoclastic bone resorption only in combination with TNF-alpha. The pathway by which the cytokine combination exerts this effect was examined using inhibitors of NO synthesis and NF-kappaB activation. Although both inhibitors used abolished NO production, they did not prevent the stimulatory effect of the cytokine combination on osteoclastic resorption. In contrast, the inhibitors slightly increased osteoclastic resorption, suggesting a suppressive rather than stimulatory effect of NO on cytokine-induced bone resorption. In addition, we showed that IL-17 + TNF-alpha stimulated osteoclastic resorption independent of NF-kappaB signaling. To further examine the pathway by which osteoclastic resorption was stimulated, we used osteoprotegerin, a specific inhibitor of the receptor activator of NF-kappaB (RANK)/receptor activator of the NF-kappaB ligand (RANKL) pathway. Osteoprotegerin partially inhibited IL-17 + TNF-alpha-stimulated osteoclastic resorption only at the high concentration of 1000 ng/mL, whereas it completely blocked parathyroid hormone-related peptide-stimulated resorption at 300 ng/mL. In conclusion, IL-17 stimulated NO production by an NF-kappaB-dependent pathway in osteoblastic cells and fetal mouse metatarsals only in combination with TNF-alpha. Neither NO production nor NF-kappaB signaling, and only partly the RANK/RANKL pathway, were involved in the stimulatory effect of the cytokine combination on osteoblastic bone resorption in these long bones, suggesting the existence of other pathways by which osteoclastic resorption can be stimulated.

Interleukin-17: A new bone acting cytokine in vitro.

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.

Oestrogenic compounds modulate cytokine-induced nitric oxide production in mouse osteoblast-like cells.

Nitric oxide (NO) is a mediator of bone metabolism with effects on both bone resorption and formation. Its production by both the constitutive and inducible isoforms of nitric oxide synthase (NOS) is affected by oestrogen in several types of cell and in tissues other than bone cells. Recently, oestrogens were found to increase basal NO production by osteoblasts via enhanced activity or expression, or both, of NOS-3. Inflammatory cytokines, however, increase NO by increasing the expression of NOS-2. In this study we have examined whether cytokine-induced NO production by osteoblastic cells was affected by oestrogenic compounds by studying the effect of 17beta-oestradiol and the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen on cytokine-induced NO production in oestrogen receptor positive MC3T3-E1 osteoblast-like cells. Combinations of the inflammatory cytokines interleukin-1beta, tumour necrosis factor-alpha, and interferon-gamma with lipopolysaccharide stimulated NO production up to 11-fold. This cytokine-induced NO production was further increased dose-dependently by the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen (133.3 +/- 3.2% and 146.0 +/- 13.2%, respectively). 17Beta-oestradiol either had no effect on or slightly inhibited cytokine-induced NO production. It did, however, dose-dependently counteract the stimulatory effect of the anti-oestrogens. Concentrations of 17beta-oestradiol needed to prevent the stimulatory effect of 4-hydroxytamoxifen were ca tenfold that of ICI164,384. These findings show that, in addition to the stimulatory effect of oestrogen on basal NO production by NOS-3, cytokine-induced NO production is also affected by oestrogenic compounds in osteoblasts.

Plasma nitrate+nitrite levels are regulated by ovarian steroids but do not correlate with trabecular bone mineral density in rats.

Nitric oxide (NO) is a mediator of bone metabolism and its production is under the control of gender hormones in several cell types or tissues. Changes in endogenous NO production, measured as plasma nitrate+nitrite levels, may therefore contribute to ovariectomy (OVX)-induced bone loss. We studied plasma nitrate+nitrite levels and trabecular bone mineral density (TBMD) 4 weeks after sham-operation or OVX in rats receiving various hormonal treatments. OVX decreased plasma nitrate+nitrite levels significantly and this was accompanied by a significant decrease in TBMD. Treatment with oral ethinyl oestradiol (EE) and subcutaneous 17beta-oestradiol dose-dependently prevented the decrease in plasma nitrate+nitrite levels after OVX, but treatment with oral 17beta-oestradiol did not. Oestrogen treatment, 17beta-oestradiol (s. c. or orally) or EE (orally), prevented the OVX-induced decrease in TBMD. Treatment of sham-operated rats with the anti-oestrogen ICI164, 384 induced a significant decrease in TBMD that corresponded to 54% of the decrease observed after OVX, but did not affect plasma nitrate+nitrite levels. Treatment of ovariectomized rats with Org 2058, a pure progestagen, did not prevent bone loss, but prevented the decrease in plasma nitrate+nitrite levels dose-dependently. Treatment with tibolone, a synthetic steroid with combined weak oestrogenic, progestagenic, and androgenic properties, or with progestagen in combination with EE completely prevented bone loss after OVX. These treatments, however, only partly prevented the OVX-induced decrease in plasma nitrate+nitrite levels. In conclusion, OVX decreased both TBMD and plasma nitrate+nitrite levels. Although plasma nitrate+nitrite levels were under the control of both oestrogen and progesterone, TBMD was affected by oestrogen only. Decreased systemic production of NO is, therefore, not involved in OVX-induced bone loss in rats.

IL-1alpha, IL-1beta, IL-6, and TNF-alpha steady-state mRNA levels analyzed by reverse transcription-competitive PCR in bone marrow of gonadectomized mice.

Loss of gonadal function in both females and males is associated with increased rates of bone loss by a yet unidentified mechanism. There is ample evidence that cytokines that are produced in the bone microenvironment and stimulate the activity and/or formation of osteoclasts are involved. In the present study, we examined whether gonadectomy increases cytokine production via increased transcription in the bone marrow of mice. For this, the in vivo steady-state mRNA levels of multiple cytokines were determined in the central bone marrow compartment of mice at different time points following ovariectomy or orchidectomy by reverse transcription-competitive polymerase chain reaction. The limit of detectable differences in mRNA expression was approximately 2-fold. Bone marrow mRNA levels of the cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) were elevated up to 30-fold after treatment of mice with lipopolysaccharide. Following gonadectomy, there were no differences in the mRNA expression of these cytokines in bone marrow of female and male mice 4, 7, and 14 days after surgery. Gender steroid deficiency does not, therefore, increase steady-state mRNA levels of IL-1alpha, IL-1beta, IL-6, and TNF-alpha in cells of the central bone marrow compartment in mice. If changes have occurred these should have been less than 2-fold or in a small cell population. These results do not preclude an important role of these cytokines in the induction of bone loss after gonadectomy. For example, bone marrow cells situated close to the bone surface or bone cells may be responsible for increased cytokine synthesis. Alternatively, the loss of gender steroids may alter post-transcriptional events in cytokine synthesis and activity or may modify the responsiveness of target cells.

Prognostic markers for survival in patients with metastatic renal cell carcinoma treated with interleukin-2.

Interleukin-2 (IL-2)-based immunotherapy can induce antitumor responses in about 25% of patients with metastatic renal cell carcinoma (RCC). The limited effect and the severe side-effects of IL-2 have led us to perform a prognostic factor analysis. Twenty-four patients with metastatic RCC were treated with IL-2. Flow cytometry and immunohistology were used to determine DNA ploidy, HLA-II expression on tumor cells, and the presence of macrophages in the primary tumor. These variables were examined in relation to survival. The 4-year overall survival rate was 38%. Forty-six percent of the primary tumors were aneuploid. All tumors, except one, showed HLA-II expression and macrophage presence. A statistically significant correlation (r = 0.66, P = 0.002) was found between HLA-II expression and macrophage presence. Patients with high HLA-II expression had a lower 4-year survival (22% compared to 50%), as had patients with high macrophage presence (20% compared to 42%). Of note, patients characterized by both high HLA-II and high macrophage expression had the worst survival (13% compared to 50%). We concluded that DNA ploidy was not predictive for survival, whereas HLA-II expression and macrophage presence may represent valuable prognostic factors related to survival. The present data suggest that more of the patients with no or moderate HLA-II expression and/or no or moderate macrophage presence in the primary tumor could survive with persistence of their malignant disease after having received IL-2 immunotherapy, as compared to patients with both high HLA-II and high macrophage expression.

Aging effects on hepatic NADPH cytochrome P450 reductase, CYP2B1&2, and polymeric immunoglobulin receptor mRNAs in male Fischer 344 rats.

Aging perturbs the expression of many liver proteins, but the mechanisms remain unresolved. Expression of hepatic NADPH cytochrome P450 reductase, phenobarbital-induced CYP2B1&2, and the polymeric immunoglobulin receptor (pIgR) decline as a function of aging. We examined the effect of aging on the expression of the mRNA transcripts of these proteins, as well as those of alpha 2u-globulin and beta-actin in male F344 rats. Despite age-related losses in the expression of P450 reductase and plasma membrane-bound pIgR in the rat liver (approximately 30-50%), aging is is accompanied by 1) no change and 2) a modest decline (< 20%) in their respective mRNA steady state levels. On the other hand, the expression of phenobarbital-induced microsomal CYP2B1&2 and the steady state level of its mRNA exhibit parallel age-dependent shifts. The mRNA transcript for alpha 2u-globulin declines between maturity and old age, whereas the beta-actin mRNA level remains unchanged. These preliminary data are consistent with previous studies which suggest that aging may perturb hepatic CYP2B1&2 and alpha 2u-globulin at the transcriptional level, whereas changes in the expression of P450 reductase and pIgR may reflect posttranscriptional modifications.