Joachim Albers

Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP+ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption.

Control of bone formation by the serpentine receptor Frizzled-9.

Fzd9, induced upon osteoblast differentiation, is required for bone matrix mineralization in primary osteoblasts.

WNT5A is induced by inflammatory mediators in bone marrow stromal cells and regulates cytokine and chemokine production

WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)–derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF‐α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF‐α transgenic (hTNFtg) mice. A microarray analysis of WNT5A‐treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS‐purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF‐α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A‐treated osteoblasts identified cytokines and chemokines as targets. The induction of IL‐1β, IL‐6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF‐κB, mitogen‐activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS‐induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A‐treated BMSC was increased by 25% and 20%, respectively. This study underlines the critical role of BMSC‐derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.

Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts

The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.

Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

Impaired bone formation and increased osteoclastogenesis in mice lacking chemokine (C-C motif) ligand 5 (Ccl5)

Chemokines play crucial roles in the recruitment of specific hematopoietic cell types, and some of them have been suggested to be involved in the regulation of bone remodeling. Because we have previously observed that chemokine (C‐C motif) ligand 2 (Ccl2) and Ccl5 are direct target genes of noncanonical Wnt signaling in osteoblasts, we analyzed the skeletal phenotypes of Ccl2‐deficient and Ccl5‐deficient mice. In line with previous studies, Ccl2‐deficient mice display a moderate reduction of osteoclastogenesis at the age of 6 months. In contrast, 6‐month‐old Ccl5‐deficient mice display osteopenia associated with decreased bone formation and increased osteoclastogenesis. Moreover, unlike in wild‐type and Ccl2‐deficient mice, large areas of their trabecular and endocortical bone surfaces are not covered by osteoblasts or bone‐lining cells, and this is associated with a severe reduction of endosteal bone formation. Although this phenotype diminishes with age, it is important that we could further identify a reduced number of osteal macrophages in 6‐month‐old Ccl5‐deficient mice, because this cell type has previously been reported to promote endosteal bone formation. Because Ccl5‐deficient mice also display increased osteoclastogenesis, we finally addressed the question of whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclastogenesis. For that purpose we isolated these cells by CD11b affinity purification from calvarial cultures and characterized them ex vivo. Here we found that they are unable to differentiate into osteoblasts or osteoclasts, but that their conditioned medium mediates an antiosteoclastogenic effect, possibly caused by interleukin‐18 (IL‐18), an inhibitor of osteoclastogenesis expressed by osteal macrophages. Taken together, our data provide in vivo evidence supporting the previously suggested role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, Ccl5‐deficient mice represent the first model with a spontaneous partial deficiency of osteal macrophages, a recently identified cell type, whose impact on bone remodeling is just beginning to be understood.

Osteolytic prostate cancer cells induce the expression of specific cytokines in bone-forming osteoblasts through a Stat3/5-dependent mechanism

Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells with bone cells results in a local activation of bone formation and/or bone resorption. Since the cellular and molecular mechanisms underlying the development of these tumor-induced osteoblastic or osteolytic lesions are still poorly understood, we have compared the effects of two prostate cancer cell lines, osteoblastic MDA-PCa-2b cells and osteolytic PC-3 cells, on bone-forming osteoblasts. Using Affymetrix Gene Chip hybridization followed by qRT-PCR confirmation we were able to identify specific genes, including Smpd3 and Dmp1, whose expression is significantly reduced upon treatment with PC-3-conditioned medium. Moreover, we observed that PC-3-conditioned medium led to a marked induction of several cytokine genes, including Cxcl5, Cxcl12 and Tnfsf11, the latter one encoding for the osteoclast differentiation factor Rankl. Likewise, when we analyzed the effects of MDA-PCa-2b- and PC-3-conditioned medium on signal transduction in osteoblasts we did not only observe opposite effects on the canonical Wnt signalling pathway, but also a specific induction of Erk and Stat phosphorylation by PC-3-conditioned medium. Most importantly, the induction of Cxcl5, Cxcl12 and Tnfsf11 in osteoblasts by PC-3-conditioned medium was abrogated by the Stat3/5 inhibitor piceatannol, whereas the selective blockade of Stat1 and Erk activation had no effect. Together with the finding, that activated Stat3 in osteoblasts was detectable in bone biopsies from patients with osteolytic metastases, our data suggest that the Stat3/5-dependent activation of cytokine expression in osteoblasts may have a significant impact on cancer cell migration and proliferation, but also on osteoclast activation.

p65-Dependent production of interleukin-1β by osteolytic prostate cancer cells causes an induction of chemokine expression in osteoblasts

Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.

Identification of molecular markers for articular cartilage.

OBJECTIVE The aim of the current study was to identify molecular markers for articular cartilage (AC) that can be used as tools for the quality control of tissue engineered (TE) cartilage. DESIGN A genome-wide expression analysis was performed using RNA isolated from articular and growth plate (GP) cartilage, both extracted from the knee joints of 6 weeks old minipigs. After confirming the specific expression for selected genes by RT-PCR, these were used as molecular markers for the quality control of TE cartilage. RESULTS Albeit several known chondrocyte markers were expressed to a similar extent in articular and GP cartilage, our genome-wide expression analysis led us to identify genes being selectively expressed in either GP or articular chondrocytes. These findings led us to perform a RT-PCR expression analysis for the corresponding genes to demonstrate the absence of GP-specific markers in TE cartilage, while common or AC markers were expressed. CONCLUSIONS Taken together, these results provide important novel insights into chondrocyte biology in general and AC in particular. In addition, it is reasonable to speculate, that some of the identified genes play distinct roles in the regulation of articular chondrocyte differentiation and/or function, thereby raising the possibility that they may serve as targets for non-operative therapies of osteoarthritis (OA).

The Lysosomal Protein Arylsulfatase B Is a Key Enzyme Involved in Skeletal Turnover: ARSB IS REQUIRED FOR BONE REMODELING

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate‐resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6‐phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan‐degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS‐VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5–/– mice, Arsb‐deficient mice display lysosomal storage accumulation in osteoclasts, impaired osteoclast activity, and high trabecular bone mass. Of note, the most prominent lysosomal storage accumulation was observed in osteocytes from Arsb‐deficient mice, yet this pathology did not impair production of sclerostin (Sost) and Fgf23. Because the influence of enzyme replacement therapy (ERT) on bone remodeling in MPS‐VI is still unknown, we additionally treated Arsb‐deficient mice by weekly injection of recombinant human ARSB from 12 to 24 weeks of age. We found that the high bone mass phenotype of Arsb‐deficient mice and the underlying bone cell deficits were fully corrected by ERT in the trabecular compartment. Taken together, our results do not only show that the function of Acp5 in osteoclasts is linked to dephosphorylation and activation of lysosomal enzymes, they also provide an important proof‐of‐principle for the feasibility of ERT to correct bone cell pathologies in lysosomal storage disorders.